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Clinical Cancer Research Vol. 11, 6050-6056, August 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Genetic Variations in the Glucocorticoid Receptor Gene Are Not Related to Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia

Wim J.E. Tissing1, Jules P.P. Meijerink1, Monique L. den Boer1, Bas Brinkhof1, Elisabeth F.C. van Rossum2, Elisabeth R. van Wering4, Jan W. Koper2, Pieter Sonneveld3 and Rob Pieters1

Authors' Affiliations: 1 Department of Pediatric Oncology/Hematology, Erasmus Medical Center/Sophia Children's Hospital; 2 Endocrinology and 3 Hematology, Erasmus Medical Center, Rotterdam, the Netherlands; and 4 Dutch Childhood Oncology Group, the Hague, the Netherlands

Requests for reprints: Jules P.P. Meijerink, Erasmus Medical Center/Sophia Children's Hospital, Rotterdam, Dr. Molewaterplein 60, 3015 GJ, Rotterdam, the Netherlands. Phone: 31-10-408-9379; Fax: 31-10-408-9433; E-mail: j.meijerink{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid sensitivity is an important prognostic factor in pediatric acute lymphoblastic leukemia (ALL). For its antileukemic effect, glucocorticoid binds the intracellular glucocorticoid receptor (GR) subsequently regulating transcription of downstream genes. We analyzed whether genetic variations within the GR gene are related to differences in the cellular response to glucocorticoids.

Methods: In leukemic samples of 57 children, the GR gene was screened for nucleotide variations using a PCR/single-strand conformational polymorphism sequencing strategy. Data were linked to in vivo and in vitro glucocorticoid resistance.

Results: No somatic mutations were detected in the GR gene coding region, but six polymorphisms (i.e., ER22/23EK, N363S, BclI, intron mutation 16 bp upstream of exon 5, H588H, and N766N) were identified. In 67% of ALL cases, at least one minor allele of these polymorphisms was detected. Although only borderline significant, the incidence for the N363S polymorphism minor allele was higher (12% versus 6%, P = 0.06) and for the ER22/23EK minor allele lower (4% versus 7.6%, P = 0.1) than in a healthy, comparable population. The different genotypes of the polymorphisms were not related to prednisone resistance. In conclusion, polymorphisms but not somatic mutations in the GR gene coding region occur in leukemic blasts of children with ALL. Our data suggest that these genetic variations are not a major contributor for differences in cellular response to glucocorticoids in childhood ALL. The higher incidence of the N363S minor allele and the lower incidence of the ER22/23EK minor allele in our ALL population as compared with a normal population warrants further research.

Glucocorticoids are the keystone in the treatment of children with acute lymphoblastic leukemia (ALL). In vivo and in vitro glucocorticoid response are important prognostic factors for outcome (1–5).

Glucocorticoids enter the cell by passive diffusion and bind to the intracellular glucocorticoid receptor (GR). The glucocorticoid-GR complex is translocated to the nucleus, where it triggers transactivation as well as transrepression of glucocorticoid-responsive genes. The transactivation and/or transrepression of downstream genes finally result in the induction of programmed cell death (apoptosis). The presence of point mutations or particular polymorphisms in the GR gene may lead to an impaired formation of the glucocorticoid-GR complex or alter the transactivation or transrepression process.

In the last decade, patients with generalized glucocorticoid resistance syndromes were described, which were linked to somatic mutations in the GR gene and which were localized within specific domains of the GR gene (Fig. 1A and B; refs. 6–10). Besides somatic mutations, polymorphisms within the GR gene have been described in healthy populations ( Table 1), of which some were associated with altered responsiveness to glucocorticoid. The ER22/23EK polymorphism results in decreased sensitivity for glucocorticoid (11), whereas two other polymorphisms (N363S and BclI) have been associated with an increased glucocorticoids sensitivity as measured with a dexamethasone suppression test (i.e., the response of the serum cortisol level upon 1 mg dexamethasone given orally the evening before) in asymptomatic healthy adults (12–14). The clinical relevance of the increased sensitivity for glucocorticoids in relation to the N363S polymorphism remains controversial (15–19), but the BclI polymorphism has been related to a higher body mass index, abdominal obesity, and higher systolic blood pressure (12, 20, 21). Besides these three polymorphisms, various other polymorphisms have been described with no or an unknown relationship to glucocorticoid responsiveness (19, 22–24).



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  		Fig. 1. Human GR gene with known mutations and polymorphisms. A, gene structure of the GR. Known somatic mutations and polymorphisms (arrows). C643R and L753F are mutations found in ALL cell lines. B, location of functional domains within the GR gene.

 

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  		Table 1. Polymorphisms in the GR as reported in the literature

 
To date, primary ALL patient cells have not been analyzed for genetic variations in the GR gene, which might explain glucocorticoid resistance. Somatic mutations in the GR gene have been described in two ALL cell lines (L753F and C643R) for which it is yet unclear whether these mutations alter the response to glucocorticoids (25–28).

In the present study, we analyzed the incidence of genetic variations in the coding region of the GR gene and the BclI restriction site and whether such genetic variations are related to in vitro or in vivo glucocorticoid resistance in childhood ALL.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. All patients were treated according to the Dutch Childhood Oncology Group ALL treatment protocols 7 and 8. These protocols start with a 7-day monotherapy of prednisone and one intrathecal dose of methotrexate. The prednisone response was determined on day 8: prednisone good response is defined as <1,000 leukemic blasts/µL peripheral blood and prednisone poor response as ≥1,000 blasts/µL (29). Patient material was taken before initial therapy. To differentiate between (leukemic) somatic mutations and polymorphisms, we also collected normal peripheral blood mononuclear cells of the same patients at complete remission. The study has been approved by the medical ethical committee of the Erasmus Medical Center Rotterdam and written informed consent was obtained from the patients and/or their parents or guardians as appropriate. The mononuclear cell fraction was separated by Lymphoprep density gradient centrifugation (density, 1.077 g/mL; Nycomed Pharma, Oslo, Norway), and when necessary, nonleukemic cells were depleted by immunomagnetic beads to purify the samples to >90% of leukemic cells.

DNA isolation. DNA was extracted using Trizol reagent (Life Technologies (Breda, the Netherlands), according to the protocol supplied by the manufacturer with some modifications. An additional protein degradation step was done using Proteinase K (1 mg/mL). DNA was purified in two separate steps. In the first step, DNA was precipitated using 0.5 mol/L NaCl, 60 µg of glycogen, and 2.5 volumes of 70% ethanol followed by an additional phenol extraction and a chloroform extraction. In the second purification step, DNA was precipitated in 0.3 mol/L NaAc and 2 volumes of ethanol 70%.

PCR amplification. One hundred nanograms of genomic DNA were used to amplify the GR gene using the primers as described in Table 2. The Genbank accession no. AC091925 was used as a reference GR sequence to which sequence variations were compared. For the exons 3 to 9-{alpha}, the primers were located in intronic DNA directly flanking these exons. Because exon 2 was too large to be amplified in a single PCR, five overlapping PCRs were developed to cover exon 2 (Table 2). The PCR reaction was done in 100 µL 1x PCR buffer II (Applied Biosystems, Foster City, CA), 3 mmol/L MgCl2 (except exon 2-4: 1.5 mmol/L), 250 nmol/L of deoxynucleotide triphosphates (10 µmol/µL, Amersham Biosciences, Freiburg, Germany), 3 units of AmpliTaq Gold (5 units/µL, Applied Biosystems), 100 ng DNA, and 400 nmol/L of the forward and reverse primers. Before amplification, DNA was denatured and AmpliTaq Gold activated for 9 minutes at 95°C followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C (except exons 2-4: 59°C) for 30 seconds, extension at 72°C for 60 seconds, and a final period of extension at 72°C for 10 minutes.


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  		Table 2.

 
Single-strand conformational polymorphism analysis. Ten microliters of PCR product were mixed with 45 µL of denaturating loading buffer. For optimal resolution, two loading buffers were used separately: buffer I contained 50 mmol/L of NaOH, 1 mmol/L of EDTA, and 0.05% bromophenol blue; buffer II contained 96% of formamide, 20 mmol/L EDTA, and 0.05% bromophenol blue. DNA was denatured in buffers I and II by heating for 10 minutes at 50°C and 95°C, respectively, followed by direct cooling on ice. Six microliters were loaded on a 12.5% polyacrylamide gel (Genegel Excel, Amersham Biosciences). The products were separated on a Genephor electrophoresis unit (Amersham Biosciences) with accompanying bufferstrips. (Amersham Biosciences), using two different temperatures for optimal resolution (5°C and 18°C). The DNA fragments were separated for 2 hours at 600 V, 25 mA, and 15 W. Gels were stained using the PlusOne DNA silver staining kit (Amersham Biosciences).

Sequencing. PCR products displaying an abnormal migration pattern on single-strand conformational polymorphism (SSCP) gels were sequenced. PCR products were purified using the QIAquick PCR purification kit. (Qiagen, Leusden, the Netherlands) The BigDye Terminator V1.1 cycle sequencing kit (Applied Biosystems) was used according to the protocol as provided by the manufacturer. Sequencing primers used were the same as used in the PCR reaction (Table 2), although for some reactions separate sequencing primers were developed (Table 2). The products of the sequencing reaction were analyzed using the ABI Prism 310 Genetic Analyzer (Applied Biosystems).

Taqman analysis of the BclI polymorphism. Because the BclI polymorphism is located in intron 2 and is not covered by exonic PCR amplification reactions, a separate analysis was done as described previously (12). Briefly, an allelic discrimination using Taqman chemistry on an ABI PRISM 7700 sequence detector (Applied Biosystems) was used. Primers and probe as depicted in Table 2 were used at concentrations of 400 and 50 nmol/L, respectively.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In vitro drug cytotoxicity was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described earlier. (2, 5, 30) Briefly, patient blasts were cultured with or without prednisolone disodiumphosphate in a concentration range between 0.06 and 250 µg/mL. At day 4, MTT is added, which can only be reduced into formazan by viable cells. The reduced product was measured spectrophotometrically at 562 nm. The leukemic cell survival is calculated by (absorbance drug-treated well / absorbance control wells without drug) x 100%. The LC50 value represents the concentration of the drug at which 50% of the cells are killed and is used as measure of in vitro drug cytotoxicity. In vitro prednisolone sensitivity was determined as sensitive (LC50 ≤ 0.1 µg/mL), intermediate (LC50 = 0.1-150 µg/mL), or resistant (LC50 ≥ 150 µg/mL), as has previously been described to be of prognostic value (5, 31).

Statistics. The {chi}2 test for trend and the Mann-Whitney test with correction for tied ranks were used to test for a difference in genotype distribution for the polymorphisms between the study population and a healthy population. A logistic regression analysis, the {chi}2 test for trend, and the Mann-Whitney test with correction for tied ranks were used to test for a relationship between the different genotypes (wild type, heterozygous, or homozygous) and in vivo or in vitro glucocorticoid resistance. A power analysis yields that with P < 0.05, a standardized difference of 0.85 in such a study (42 in vivo prednisone sensitive and 15 resistant) is detected with 0.8 power. This allows to draw conclusions on large differences between sensitive and resistant patients, which is what we expect when glucocorticoid resistance is determined by genetic variations. The probability of event-free survival was calculated using the Kaplan-Meier method and a correlation between different genotypes and probability of event-free survival was analyzed using the log-rank test.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In this study, 57 children with ALL were included for which in vitro and/or in vivo response towards glucocorticoid was known. (Patient characteristics are given in Table 3) To study whether in vitro or in vivo resistance correlated with the presence of genetic variations within the GR gene, we have developed a PCR/SSCP strategy. The sensitivity of this SSCP technique varies between 80% and 95% using a single SSCP condition (32). In the current study, we used two denaturating conditions and two temperature conditions to further improve the level of detection of genetic variations in the GR. PCR products showing an abnormal migration pattern on SSCP gel were sequenced. To check for the sensitivity and specificity of the assay, control samples with known genotype (wild type, heterozygote, and homozygote) for the polymorphisms N363S, the intron mutation upstream of exon 5, and N766N, obtained from a previous study of Koper et al. (22), were analyzed and successfully distinguished (data not shown). Second, in all PCR products with an aberrant SSCP pattern, a nucleotide variation was found after DNA sequencing. Third, 50 PCR products with a normal SSCP pattern were sequenced, showing only wild-type alleles.


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  		Table 3. Patient characteristics of the study population

 
Each genetic variation had its specific denaturating buffer and temperature at which the resolution was most optimal. For example, N363S was detected with both buffers I and II at 18°C, whereas the intron mutation 16 bp upstream of exon 5 only was detected with buffer II at 5°C. Figure 2A shows an example of the SSCP pattern for the PCR products of exon 5 for 13 patients. The results of the sequencing procedure for three of these patients (wild type, heterozygote, and homozygote) are shown in Fig. 2B.



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  		Fig. 2. Example of the detection of mutations in the GR by PCR-SSCP and sequence analysis. A, SSCP pattern of exon 5 of 12 patient samples, representing an intron mutation 16 bp upstream of exon 5: wild type (lanes 2, 3, 7, 11, and 12), heterozygous (lanes 1, 4, 5, 6, 9, 10, and 13), and homozygous mutation (lane 8). B, sequence analysis of three patients for the site of the intron mutation 16 bp upstream of exon 5. Wild-type patient (1), heterozygous (2), and homozygous patient (3).

 
SSCP analysis of the coding exons 2 to 9 of the GR gene in 57 patients and subsequent DNA sequencing of aberrant SSCP patterns resulted into the detection of six different genetic variations. These six genetic variations were all identified as previously known polymorphisms (i.e., ER22/23EK, N363S, BclI, intron mutation 16 bp upstream of exon 5, H588H, and N766N; Table 3). No previously unknown mutations were detected in these patients. In 55% of the patients with one or more GR gene sequence alterations in the ALL blasts, normal mononuclear cells at the time of complete remission were available for further analysis. In all cases, the sequence alterations such as found in the leukemic blasts were also present in these nonmalignant cells, indicating that these alterations were indeed polymorphisms. The genotype distribution was consistent with a Hardy-Weinberg equilibrium.

In 38 of 57 patients (67%), at least one minor allele of the polymorphisms was found. In two patients we found four minor alleles, in 11 patients three minor alleles, in nine patients two minor alleles, and in 16 patients one minor allele. In 19 patients, no abnormalities were detected. (Note that only 53 patients were screened for the BclI polymorphism and only 56 patients were screened for the N766N polymorphism).

The genotype distribution of the polymorphisms in our cohort was comparable to the incidence as reported in literature, except for the N363S and ER22/23EK polymorphisms (Fig. 1C). The minor allele of the N363S polymorphism was observed in a higher percentage of ALL patients (12%) and the minor allele of ER22/23EK polymorphism was found in only 4% of the patients as compared with 6% and 7.4% respectively in the normal, healthy population from the same topographical region. The test to compare the incidence in the study population and the incidence as reported in the literature yielded borderline significance (P = 0.06 and P = 0.1).

The relationship between the polymorphisms and in vivo and in vitro glucocorticoid resistance is shown in Table 3. We analyzed a possible relationship between glucocorticoid resistance versus wild type, heterozygote, and homozygote as well as a possible relationship between glucocorticoid resistance versus wild type and one minor allele (i.e., heterozygote or homozygote). None of the polymorphisms seemed correlated with in vivo prednisone response (n = 57) nor with in vitro prednisolone resistance (n = 40; Table 4). The presence of two minor alleles in one patient (e.g., N363S and BclI) did not correlate with in vivo or in vitro glucocorticoid resistance as well. In this small cohort of 57 patients, we did not find an association between the presence of polymorphisms and event-free survival (data not shown).


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  		Table 4. Genetic variations in the GR gene, incidence and relation to in vivo prednisone and in vitro prednisolone sensitivity

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids are important drugs in the multidrug treatment protocols for children with ALL. The in vivo prednisone response as used in the BFM studies has been found to be one of the strongest prognostic factors for therapy outcome in childhood ALL (1, 29, 33). The in vitro prednisolone sensitivity of untreated leukemic cells, as determined by the MTT assay, is a strong risk factor for outcome as well and can predict the occurrence of a relapse even within the group of patients with a good in vivo prednisone response (2, 4, 5, 31).

To induce apoptosis in glucocorticoid-sensitive cells, glucocorticoids have to bind the intracellular GR. This results in transactivation and/or transrepression of glucocorticoid-responding genes, which eventually results in apoptosis in glucocorticoid-sensitive patients. Although glucocorticoid resistance is an important cause of treatment failure, little is known about possible mechanisms of glucocorticoid resistance in childhood ALL (34). In this study, we tested the hypothesis that glucocorticoid resistance is the result of genetic variations within the GR gene, which might alter the glucocorticoid-binding capacity or transactivation or transrepression of glucocorticoid responding genes.

Analysis of the coding region of the GR gene for sequence alterations in 57 patients revealed six different genetic variations. These genetic variations were identical to previously reported polymorphisms (i.e., ER22/23EK, N363S, BclI, intron mutation 16 bp upstream of exon 5, H588H, and N766N; Fig. 1). No somatic mutations that might result in altered glucocorticoid sensitivity were found in these 57 ALL cases. In two studies, somatic mutations were found in cell lines from ALL patients. A L753F somatic mutation was shown to be present in CCRF-CEM cells and original ALL cells from which the cell line was established (27). As the L753F somatic mutation was found in both the glucocorticoid sensitive and resistant clones of the CCRF-CEM cell line, it probably has no correlation with glucocorticoid sensitivity (25, 26). The C643R somatic mutation was found in a cell line established from a Japanese patient with ALL (P30/OHK). It is not clear whether this mutation was also present in the original cells or that it was acquired during tissue culture. Transfection studies showed that the C643R allele had no transcriptional activity, but no evidence was given whether this mutation was related to glucocorticoid resistance in ALL cells (28). All together, these data suggest that somatic mutations do not occur frequently and can not explain resistance to glucocorticoid in pediatric ALL. In contrast to somatic mutations, we detected several polymorphisms of the GR gene in children with ALL. Confirmation of the same genetic variations in normal peripheral blood mononuclear cells obtained during clinical remission confirmed that the variations indeed were polymorphisms. Other polymorphisms of the GR gene as reported in the literature with a low incidence were not detected in our population nor did we detect any thus far unreported genetic variations. Although we can not exclude that these polymorphisms with low incidence were missed by our PCR/SSCP, we had optimized our strategy to achieve a high detection limit; thus, it is reasonable to assume that these low-incidence polymorphisms were absent in our population.

The genotype distribution of the polymorphisms BclI, N766N, and the intron mutation upstream of exon 5 was the same as reported in the literature. However, the minor allele of the ER22/23EK polymorphism was found in a lower percentage than in a healthy population of the same ethnicity (4% versus 7.4%, respectively), whereas the minor allele of N363S was found in a higher percentage as compared with the healthy population of the same ethnicity (12% versus 6%, respectively; ref. 13). Because the number of patients in our study is small and no statistical significance was reached (P = 0.1 and P = 0.06, respectively), the altered frequency of both polymorphisms warrants further evaluation in a larger cohort of patients. The minor allele of the H588H polymorphism was found in one of our patients. It was reported before in cell lines that originate from four Japanese individuals (24). In a previous Dutch study including a large healthy population, this polymorphism was not reported (22).

In the literature, the clinically most relevant polymorphism of the GR gene is the BclI polymorphism (12). Previously, it was detected with a RFLP-based technique, but recently, the exact mutation was found to be a C/G single nucleotide polymorphism in intron 2, 646 bp downstream from exon 2 for which a specific real-time PCR approach has been developed (12, 35). It is associated with increased response to a dexamethasone suppression test and with increased (abdominal) obesity, body mass index, and blood pressure (20, 36, 37). However, in our population, the BclI polymorphism was not correlated with in vivo or in vitro glucocorticoid sensitivity. In addition, for the other five polymorphisms, no correlation with in vivo or in vitro glucocorticoid response was found.

In conclusion, polymorphisms but not somatic mutations in the coding region of the GR gene occur in leukemic blasts of children with ALL. Our data suggest that genetic variations in the GR gene are not a major contributor for differences in cellular response to glucocorticoid in childhood ALL.


   Acknowledgments
 
We thank Dr. H.B. Beverloo (Department of Clinical Genetics, Erasmus Medical Center, Rotterdam) for supplying DNA from leukemic blasts; Prof. J.J.M. van Dongen (Department of Immunology, Erasmus Medical Center) for providing us with DNA from patients in continuous complete remission; and Dr. R.X. Menezes for statistical support.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/14/04; revised 5/23/05; accepted 5/27/05.


   References
Top
 Abstract
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 Results
 Discussion
 References
 

  1. Lauten M, Stanulla M, Zimmermann M, Welte K, Riehm H, Schrappe M. Clinical outcome of patients with childhood acute lymphoblastic leukaemia and an initial leukaemic blood blast count of less than 1000 per microliter. Klin Padiatr 2001;213:169–74.[CrossRef][Medline]
  2. Kaspers GJ, Pieters R, Van Zantwijk CH, Van Wering ER, Van Der Does-Van Den Berg A, Veerman AJ. Prednisolone resistance in childhood acute lymphoblastic leukemia: vitro-vivo correlations and cross-resistance to other drugs. Blood 1998;92:259–66.[Abstract/Free Full Text]
  3. Pieters R, den Boer ML, Durian M, et al. Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia–implications for treatment of infants. Leukemia 1998;12:1344–8.[CrossRef][Medline]
  4. Hongo T, Yajima S, Sakurai M, Horikoshi Y, Hanada R. In vitro drug sensitivity testing can predict induction failure and early relapse of childhood acute lymphoblastic leukemia. Blood 1997;89:2959–65.[Abstract/Free Full Text]
  5. Den Boer ML, Harms DO, Pieters R, et al. Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia. J Clin Oncol 2003;21:3262–8.[Abstract/Free Full Text]
  6. Karl M, Lamberts SWJ, Detera-Wadleigh SD, et al. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 1993;76:683–9.[Abstract]
  7. Karl M, Lamberts SW, Koper JW, et al. Cushing's disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians 1996;108:296–307.[Medline]
  8. Malchoff DM, Brufsky A, Reardon G, et al. A mutation of the glucocorticoid receptor in primary cortisol resistance. J Clin Invest 1993;91:1918–25.
  9. Hurley DM, Accili D, Stratakis CA, et al. Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest 1991;87:680–6.
  10. Ruiz M, Lind U, Gafvels M, et al. Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance. Clin Endocrinol 2001;55:363–71.[CrossRef][Medline]
  11. 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;51:3128–34.[Abstract/Free Full Text]
  12. 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 (Oxf) 2003;59:585–92.[CrossRef][Medline]
  13. 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;83:144–51.[Abstract/Free Full Text]
  14. 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;59:333–57.[Abstract/Free Full Text]
  15. Rosmond R, Bouchard C, Bjorntorp P. Tsp509I polymorphism in exon 2 of the glucocorticoid receptor gene in relation to obesity and cortisol secretion: cohort study. BMJ 2001;322:652–3.[Free Full Text]
  16. Dobson MG, Redfern CP, Unwin N, Weaver JU. The N363S polymorphism of the glucocorticoid receptor: potential contribution to central obesity in men and lack of association with other risk factors for coronary heart disease and diabetes mellitus. J Clin Endocrinol Metab 2001;86:2270–4.[Abstract/Free Full Text]
  17. Di Blasio AM, van Rossum EF, Maestrini S, et al. The relation between two polymorphisms in the glucocorticoid receptor gene and body mass index, blood pressure and cholesterol in obese patients. Clin Endocrinol (Oxf) 2003;59:68–74.[CrossRef][Medline]
  18. Lin RCY, Wang WYS, Morris BJ. High penetrance, overweight, and glucocorticoid receptor variant: case-control study. BMJ 1999;319:1337–8.[Free Full Text]
  19. Lin RC, Wang WY, Morris BJ. Association and linkage analyses of glucocorticoid receptor gene markers in essential hypertension. Hypertension 1999;34:1186–92.[Abstract/Free Full Text]
  20. Rosmond R, Chagnon YC, Holm G, et al. A glucocorticoid receptor gene marker is associated with abdominal obesity, leptin, and dysregulation of the hypothalamic-pituitary-adrenal axis. Obes Res 2000;8:211–8.[Medline]
  21. Clement K, Philippi A, Jury C, et al. Candidate gene approach of familial morbid obesity: linkage analysis of the glucocorticoid receptor gene. Int J Obes Relat Metab Disord 1996;20:507–12.[Medline]
  22. 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;99:663–8.[CrossRef][Medline]
  23. Feng J, Zheng J, Bennett WP, et al. Five missense variants in the amino-terminal domain of the glucocorticoid receptor: no association with puerperal psychosis or schizophrenia. Am J Med Genet 2000;96:412–7.[CrossRef][Medline]
  24. Nagano M, Nakamura T, Ozawa S, Maekawa K, Saito Y, Sawada J. Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms. J Biochem Biophys Methods 2003;55:1–9.[CrossRef][Medline]
  25. Strasser-Wozak EMC, Hattmannstorfer R, Hala M, et al. splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line. Cancer Res 1995;55:348–53.[Abstract/Free Full Text]
  26. Powers JH, Hillmann AG, Tang DC, Harmon JM. Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells. Cancer Res 1993;53:4059–65.[Abstract/Free Full Text]
  27. Hillmann AG, Ramdas J, Multanen K, Norman MR, Harmon JM. Glucocorticoid receptor gene mutations in leukemic cells acquired in vitro and in vivo. Cancer Res 2000;60:2056–62.[Abstract/Free Full Text]
  28. Nagano M, Nakamura T, Niimi S, et al. Substitution of arginine for cysteine 643 of the glucocorticoid receptor reduces its steroid-binding affinity and transcriptional activity. Cancer Lett 2002;181:109–14.[Medline]
  29. Riehm H, Reiter A, Schrappe M, et al. Die corticosteroid-abhangige dezemierung der leukamiezellzahl im blut als prognose-faktor bei der akuten lymphoblastischen leukemie im kindesalter (therapiestudie ALL-BFM 83). Klin Padiatr 1986;199:151–60.
  30. Pieters R, Loonen AH, Huismans DR, et al. In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions. Blood 1990;76:2327–36.[Abstract/Free Full Text]
  31. Pieters R, Huismans DR, Loonen AH, et al. Relation of cellular drug resistance to long-term clinical outcome in childhood acute lymphoblastic leukaemia. Lancet 1991;338:399–403.[CrossRef][Medline]
  32. Vidal-Puig A, Moller DE. Comparative sensitivity of alternative single-strand conformation polymorphism (SCP) methods [Technical Brief]. Biotechniques 1994;17:490–6.[Medline]
  33. Schrappe M, Reiter A, Zimmermann M, et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster. Leukemia 2000;14:2205–22.[CrossRef][Medline]
  34. Tissing WJ, Meijerink JP, den Boer ML, Pieters R. Molecular determinants of glucocorticoid sensitivity and resistance in acute lymphoblastic leukemia. Leukemia 2003;17:17–25.[CrossRef][Medline]
  35. Fleury I, Beaulieu P, Primeau M, Labuda D, Sinnett D, Krajinovic M. Characterization of the BclI polymorphism in the glucocorticoid receptor gene. Clin Chem 2003;49:1528–31.[Free Full Text]
  36. Buemann B, Vohl MC, Chagnon M, et al. Abdominal visceral fat is associated with a BclI restriction fragment length polymorphism at the glucocorticoid receptor gene locus. Obes Res 1997;5:186–92.[Medline]
  37. Ukkola O, Rosmond R, Tremblay A, Bouchard C. Glucocorticoid receptor BclI variant is associated with an increased atherogenic profile in response to long-term overfeeding. Atherosclerosis 2001;157:221–4.[CrossRef][Medline]



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