JP2003265184A - Method for examining effectiveness of glucocorticoid preparation in examinee - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for examining a glucocorticoid preparation in an examinee and to obtain a test reagent. <P>SOLUTION: In a glucocorticoid receptor (GR), CR which has 643 amino acid residue substituting cystine with arginine, amino acid residues from 722 leucine to 777 lysine substituting with other amino acid residues is found ([C643R variation type GR] and [2314 ins-a variation type GR], respectively). The transcriptive activity of the C643R variation type GR and the 2314 ins-a variation type GR prove to be controlled. The bond affinity of the C643R variation type GR for dexamethasone proves to be low. Consequently, the effectiveness of glucocorticoid is predicted by examining these variations. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for examining the effectiveness of a glucocorticoid preparation in a subject.

[0002]

Glucocorticoid receptor (GR) is a transcription factor activated by glucocorticoid. GR is
First present as a cytoplasmic protein associated with some heat shock proteins and other proteins
(WB Pratt, DO Toft, Steroid receptor interac
tions with heat shock protein and immunophilin cha
perones. Endocr. Rev. 18 (1997) 306-360). Glucocorticoids bind GR in the cytoplasm, which induces GR nuclear translocation (JC Webster, JA Cidlowski,
Mechanisms of Glucocorticoid-receptor-mediated Re
pression of Gene Expression. Trends Endocrinol. Me
tab. 10 (1999) 396-402). The induction or reduction of its transcriptional activity depends on the tissue or cell type and is therefore associated with diverse and sometimes conflicting physiological effects on different tissues. That is, GR is known to be involved in glucocorticoid-induced apoptosis in lymphocytes (AH Wyllie, Glucocorticoid-indu
ced thymocyte apoptosis is associated with endogen
ous endonuclease activation. Nature 284 (1980) 555
-556; R. Schwartzman, J. Cidlowski, Glucocorticoid
-induced apoptosis of lymphoid cells. Int. Arch. Al
lergy Immunol. 105 (1994) 347-354). However, human mammary epithelial cells (MEC) are rather protected from apoptosis by GR activation (TJ Moran, S. Gray, CA Mik.
osz, SD Conzen.The glucocorticoidreceptor mediat
es a survival signal in human mammary epithelial c
ells. Cancer Res. 60 (2000) 867-72). Recently, susceptibility of lymphoid cells to glucocorticoid-dependent apoptosis was shown to be strongly dependent on the state of target cell differentiation and the presence or absence of foreign cell supporting factors (EB Thompson, Mechanisms of
T-cell Apoptosis Induced by Glucocorticoids. Trend
s Endocrinol Metab. 10 (1999) 353-358.). Basal levels of various protein kinase C (PKC), NF-κB and PKA pathways were considered as determinants of the intrinsic susceptibility of lymphoid cells to glucocorticoid-dependent apoptosis. However, very little is known about the mechanisms of these GR-mediated effects.

Glucocorticoids are one of the most potent substances used in the treatment of childhood acute lymphoblastic leukemia and are included in all standard induction treatments (C.-.
H. Pui, Childhood leukemias. N. Engl. J. Med. 332
(1995) 1618-1630; GJLKaspers, R. Pieters, E.
Klumper, FC DeWaal, AJP Veerman, Glucocort
icoid resistance in childhood leukemia. Leukemia L
ymphoma 13 (1994) 187-201). Previous studies have shown that human leukemic CEM-C1 cells are glucocorticoid resistant,
And has a heterozygous mutation (L753F) in the ligand binding domain (LBD) of the GR gene (M. Hanad
a, M. Shimoyama, Potential limitation of growth-in
hibitory action of recombinant human tumor necrosi
s factor (PAC-4D) due to easy induction of resista
nce: evidence in vitro.Jpn.J. Cancer Res. 78 (198
7) 1266-1273; EM Strasser-Wozak, R. Hattmannsto
rfer, M. Hala, BL Hartmann, M. Fiegl, S. Geley,
R. Kofler, Splice site mutation in the glucocorti
coid receptor gene causes resistance to glucocorti
coid-induced apoptosis in a human acute leukemic c
ell line. CancerRes. 55 (1995) 348-353; AG Hill
mann, J. Ramdas, K. Multanen, MR Norman, JM
Harmon, Glucocorticoid receptor gene mutations in
leukemiccells acquired in vitro and in vivo.Cance
r Res. 60 (2000) 2056-2062; S. Geley, BL Hartman
n, M. Hala, EM Strasser-Wozak, K. Kapelari, R.
Kofler, Resistance to glucocorticoid-induced apopt
osis in human T-cell acute lymphoblastic leukemia
CEM-C1 cells is due to insufficient glucocorticoid
receptor expression. Cancer Res. 56 (1996) 5033-5
038). In these cells, glucocorticoid binding affinity and transcriptional activation were markedly reduced. These reports suggest that the mutation L753F affects glucocorticoid resistance in CEM-C1 cells and that this mutation may inhibit glucocorticoid-induced apoptosis. Therefore, it was suggested that the glucocorticoid resistance of CEM-C1 cells was due to the threshold expression of functional GR rather than the defect of the signaling pathway distal to GR.

[0004] If the relationship between the mutation in the glucocorticoid gene and the decrease in GR glucocorticoid-binding ability is revealed, it is possible to evaluate the efficacy of the glucocorticoid preparation by detecting the mutation in the GR gene. Expected to be.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and an object thereof is to provide a method for examining the effectiveness of a glucocorticoid preparation in a subject. Furthermore, it aims at providing the reagent for testing the effectiveness of a glucocorticoid formulation.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above problems. First, Japanese 10
Sequence of the GR gene coding region of each individual leukemia cell line and one Japanese colon cancer cell line
We examined whether certain mutations were present in the gene. As a result, in the GR gene, a mutation C643R that replaces cysteine-643 with arginine and a mutation 2314 ins-a that replaces leucine-772 with lysine-777 with another 26 amino acids were found.

[0007] In order to examine the transcriptional activity of C643R mutant GR and 2314 ins-a mutant GR, the present inventors have examined wild type GR plasmid, C643R mutant GR plasmid or 2314 ins-a mutant GR. The plasmid was transfected into COS-7 cells using the mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc). As a result of the examination, the luciferase activity increased in a dose-dependent manner in the cells transfected with wild-type GR. However, the C643R mutant GR did not induce expression of the dexamethasone-responsive luciferase reporter gene. Similar results were obtained with the 2314 ins-a mutant GR. Therefore, it was revealed that the transcriptional activities of C643R mutant GR and 2314 ins-a mutant GR were suppressed.

Furthermore, the present inventors have investigated the affinity of C643R mutant GR for dexamethasone by using Scatcha.
According to rd plot analysis, the binding affinity to [ ³ H] dexamethasone in the cytosolic fraction of COS-7 cells transfected with the wild type or C643R mutant GR plasmid was measured. As a result, specific high affinity binding was
It was revealed in the cytoplasmic fraction prepared from COS-7 cells transfected with GR. This binding capacity is 864fmo
It was 1 / mg cytoplasmic protein, and the dissociation constant Kd was 7.4 nM. COS-7 transfected with C643R mutant GR
The binding capacity of the cytoplasmic fraction from the cells (921 fmol / mg cytoplasmic protein) was similar to that of the wild type, but the Kd value (44.6n
M) was 6 times higher. Therefore, it was revealed that the binding affinity of C643R mutant GR to dexamethasone was low.

[0009] As described above, the present inventors have found that the mutation C643R in which GR cysteine-643 is replaced with arginine, or the mutation 2314 ins-a in which leucine-772 is replaced with lysine-777 with another 26 amino acids is glucocorticoid. The present invention was completed by demonstrating for the first time that the binding ability was reduced or the transcriptional activation ability of GR was reduced. By detecting a GR gene polymorphism that reduces the glucocorticoid binding ability of GR or reduces the transcriptional activation ability of GR, it becomes possible to determine the efficacy of a glucocorticoid preparation in a subject. It was

That is, the present invention relates to a method for examining the effectiveness of a glucocorticoid preparation in a subject, and a reagent for examining the effectiveness of the glucocorticoid preparation,
More specifically, [1] a method for examining the efficacy of a glucocorticoid preparation in a subject, which comprises decreasing the glucocorticoid-binding ability of a glucocorticoid receptor or the transcription activation ability of the receptor. A method of detecting a polymorphism of a glucocorticoid receptor gene to be reduced, wherein the test method determines that the subject is resistant to the glucocorticoid preparation when the polymorphism is detected, [2] The method according to [1], wherein the type is a single nucleotide polymorphism, [3] the polymorphism in the glucocorticoid receptor gene causes a mutation at the 643th amino acid in the glucocorticoid receptor, [1] [4] The polymorphism of the glucocorticoid receptor gene, wherein the polymorphism of the glucocorticoid receptor gene results in a mutation at the 643th amino acid in the glucocorticoid receptor, and Receptor 772-777
The test method according to [1], which is a polymorphism that causes the th amino acid mutation, [5] 6 of the glucocorticoid receptor
The inspection method according to [3] or [4], wherein the 43rd amino acid mutation is a mutation from cysteine to arginine, [6] a polymorphism of the receptor gene that causes the 643th amino acid mutation of the glucocorticoid receptor. The test method according to [3] or [4], wherein the type is a polymorphism at the 1927th nucleotide site in the cDNA sequence of the gene,
[7] The test method according to [6], wherein the polymorphism at the 1927th nucleotide site in the cDNA sequence of the glucocorticoid receptor gene is a mutation from thymine to cytosine.
[8] A polymorphism in the receptor gene that causes a mutation at the 772nd to 777th amino acids in the glucocorticoid receptor is
The test method according to [4], which is a polymorphism at the 2314th base site in the cDNA sequence of the gene,

[9] The test method according to [8], wherein the polymorphism at the 2314th base site in the cDNA sequence of the glucocorticoid receptor gene is a single-base insertion mutation in the site, [10] the glucocorticoid-binding ability is , Dexamethasone binding ability, [1]-

The inspection method according to any one of [9], [1
1] Including the following steps (a) to (d), [1] to
The inspection method according to any one of [10], (a) the step of preparing a DNA sample from a subject, (b) the base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene.
Step (c) of isolating DNA containing the 927th base site, or containing the 1927th and 2314th base sites (c) Step of determining the base sequence of the isolated DNA (d) DNA determined by the step (c) The base sequence of
Step of comparing with control [12] Including the following steps (a) to (d), [1]
To (10), (a) a step of preparing a DNA sample from a subject (b) a step of cleaving the prepared DNA sample with a restriction enzyme (c) a size of the DNA fragment Step of separating according to (d) step of comparing the size of the detected DNA fragment with a control [13] including the following steps (a) to (e), [1]
To (10), (a) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
Amplifying DNA containing the 927th base site or containing the 1927th and 2314th base sites (c) Cleaving the amplified DNA with a restriction enzyme (d) Separation of DNA fragments according to their size Step (e) Comparing the size of the detected DNA fragment with a control [14] The following steps (a) to (e) are included: [1]
To (10), (a) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
A step of amplifying DNA containing the 927th base site, or containing the 1927th and 2314th base sites (c) Dissociating the amplified DNA into single-stranded DNAs (d) Dissociating the single-stranded DNAs Step of separating on non-denaturing gel (e) Step of comparing mobility of separated single-stranded DNA on gel with control [15] Including steps (a) to (d) below [1]
To (10), (a) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
Amplifying DNA containing the 927th base site or containing the 1927th and 2314th base sites (c) Separation of the amplified DNA on a gel in which the concentration of the DNA denaturant is gradually increased (d) Separation Comparing the mobility of the prepared DNA on a gel with a control [16] including the following steps (a) to (c): [1]
To (10), (a) (i) a base site in the glucocorticoid receptor gene prepared from a subject, which is the 1927th base site in the cDNA sequence of the receptor gene Or (ii) a substrate having a nucleotide probe immobilized thereon, and (ii) the DNA of step (a) (i) and step (a) ( ii)
Contacting the substrate of (c) by detecting the intensity of hybridization between the DNA and the nucleotide probe immobilized on the substrate,
A step of detecting a polymorphism in a gene encoding a glucocorticoid receptor [17] a polymorphic site in the glucocorticoid receptor gene, comprising at least the 1927th polymorphic site in the cDNA sequence of the receptor gene, A polynucleotide having a chain length of nucleotides [18]
[19] The polynucleotide according to [17], wherein the base of the polymorphic site is cytosine (C), and [19] a base site in the glucocorticoid receptor gene, wherein the 1927th base site in the cDNA sequence of the receptor gene is A reagent for testing the efficacy of a glucocorticoid preparation, which comprises a polynucleotide that hybridizes to DNA containing and has a chain length of at least 15 nucleotides, [20].
Furthermore, a polynucleotide that hybridizes to a DNA that is a base site in the glucocorticoid receptor gene and includes the 2314th base site in the cDNA sequence of the receptor gene, and has a chain length of at least 15 nucleotides is included [19 ]] [21]
A base site in the glucocorticoid receptor gene, which is 192 in the cDNA sequence of the receptor gene
A reagent for testing the effectiveness of a glucocorticoid preparation, comprising a forward primer designed to amplify a DNA containing the 7th base site, and a reverse primer, [22] further, a base in the glucocorticoid receptor gene D, which includes the 2314th base site in the cDNA sequence of the receptor gene
The reagent according to [21], which comprises a forward primer and a reverse primer designed to amplify NA.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have found an amino acid mutation in GR that reduces the glucocorticoid-binding ability of the glucocorticoid receptor (GR) or reduces the transcriptional activation capability of GR. More specifically, the inventors have found that the glucocorticoid receptor (GR) gene (Ge
In the cDNA sequence of nBank Accession No .: NM 000176), the 1927th base T was replaced by C, or the A was inserted between the 2313th base and the 2314th base. The amino acid residue of is replaced with an amino acid residue from cysteine to arginine, or
Amino acid residues from leucine-772 to lysine-777 are different
We found GR substituted with 26 amino acid residues (respectively,
Described as "C643R mutant GR" and "2314 ins-a mutant GR"). The C643R mutant GR and the 2314 ins-a mutant G
The transcriptional activity of R is suppressed, and the C643R mutant
It was found that GR has a low binding affinity for glucocorticoids. Therefore, by testing for the above mutation, it is possible to predict the efficacy of the glucocorticoid preparation before administering the glucocorticoid preparation. The present invention is based on such knowledge. In the present invention, wild-type GR gene, C643R mutant G
The cDNA sequences of the R gene and the 2314 ins-a mutant GR gene are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively. The amino acid sequences of wild-type GR, C643R mutant GR and 2314 ins-a mutant GR are shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, respectively.

[0012] The present invention is a method for testing the efficacy of a glucocorticoid preparation in a subject, which comprises reducing the glucocorticoid binding ability of GR or reducing the transcription activation ability of GR. Provided is a method for detecting a polymorphism, the method including the step of detecting the type, wherein the subject is determined to be resistant to the glucocorticoid preparation when the polymorphism is detected.

In the present invention, the glucocorticoid receptor (GR) is a receptor belonging to the nuclear superfamily having glucocorticoid as a ligand, and is a transcriptional regulatory factor that promotes or suppresses transcription in a ligand-dependent manner. .

[0014] A polymorphism is genetically defined as a change in a certain base in one gene that is present at a frequency of 1% or more in the population. However, the “polymorphism” of the present invention is not limited to this definition, and even a base change of less than 1% is included in the “polymorphism” of the present invention. Examples of polymorphisms in the present invention include, but are not limited to, polymorphisms in which several tens of nucleotides are deleted, substituted or inserted from single nucleotide polymorphism (SNP).

Examples of polymorphisms in the present invention include GR
It is a polymorphism that causes the 643rd amino acid mutation (preferably cysteine to arginine mutation), and specifically shows the 1927th base mutation (preferably thymine to cytosine mutation) in the cDNA sequence of the GR gene. However, the mutation is not necessarily limited to the above-mentioned mutation as long as it is a mutation that causes the 643rd amino acid mutation in GR. In addition, examples of the polymorphism of the present invention include a polymorphism that causes an amino acid mutation at positions 772 to 777 of GR (preferably a substitution mutation with another amino acid residue). In particular
2314th base mutation in the cDNA sequence of the GR gene (preferably a single base insertion mutation between 2313th and 2314th bases, more preferably an adenine insertion mutation between 2313th and 2314th bases) As can be shown, wild type GR
Is not particularly limited to the above-mentioned mutations as long as it is a mutation on the GR gene that allows deletion of the 772nd to 777th amino acids or substitution of another amino acid. In the present invention, in addition to the above polymorphism, another polymorphism (gene mutation) on the GR gene
May exist.

In the present invention, the efficacy of the glucocorticoid preparation is tested by detecting a polymorphism that causes the amino acid mutation at the 643rd amino acid in GR or a polymorphism that causes the amino acid mutations at the 772nd to 777th amino acids in GR. However, in a more preferred embodiment, the detection of both the polymorphism that causes the amino acid mutation at the 643rd amino acid and the polymorphism that causes the amino acid mutation at the 772nd to 777th amino acids in GR is performed with higher accuracy, and thus the efficacy of the glucocorticoid formulation Sexual examination is possible.

In the present invention, the "glucocorticoid-binding ability" refers to the binding activity between GR and glucocorticoid. This binding activity can be performed by those skilled in the art by a general method capable of measuring the binding activity between a receptor and a ligand. For example, the binding activity can be measured by examining the binding activity between GR and dexamethasone. Dexamethasone is a kind of glucocorticoid, and is a compound generally used for treating inflammation such as dermatitis and vasculitis, lymphoma, leukemia and the like. The binding activity with dexamethasone can be carried out, for example, by performing a Scatchard plot analysis of the binding affinity for ³ H-dexamethasone in the cytoplasmic fraction of COS-7 cells transfected with the GR gene to be tested. More specifically, it can be carried out by the method described in Examples below, but the method is not particularly limited.

The transcription activation ability of GR can be measured by those skilled in the art by a general reporter assay. For example, it can be carried out by introducing a reporter gene having a binding sequence for GR into cells and measuring the amount of the reporter product expressed by the binding of GR. More specifically, it can be carried out by the method shown in Examples described later.

In the present invention, the glucocorticoid preparation means a pharmaceutical composition containing glucocorticoid as an active ingredient. Usually, the glucocorticoid preparation has a pharmacological action such as inflammation suppression and leukemia cell death.
The mechanism of action of the preparation is that it first binds to GR in the cytoplasm,
The GR bound by the glucocorticoid preparation then binds to the upstream region of the gene or other transcription factor protein.
As a result, transcriptional activation or transcriptional repression of the gene group occurs to bring about a pharmacological action.

In the present invention, "resistant to a glucocorticoid preparation" means a state in which the pharmacological effect of the glucocorticoid preparation is not observed or suppressed in a subject. In the present invention, “suppression” is not limited to complete suppression, and is understood to mean “suppression” as long as it is suppressed compared to a control (usually a healthy person). For example, by the test method of the present invention, when it is determined that a certain subject is resistant to a glucocorticoid formulation, the therapeutic effect of administration of the glucocorticoid formulation to the subject is not observed, Or it can be expected to be low. Based on the information on the efficacy of the glucocorticoid preparation for the subject obtained by the method of the present invention, it is possible to appropriately determine the treatment policy and the like for the subject.

In the test method of the present invention, the polymorphism generated in the GR gene can be detected, for example, by directly determining the base sequence of the GR gene of the subject. In this method, first, a DNA sample is prepared from a subject. DNA
The sample can be prepared, for example, based on chromosomal DNA or RNA extracted from peripheral blood leukocytes, tissues or cells such as skin, oral mucous membranes, tears, saliva, urine, feces or hair of a subject.

In the present method, next, the base site in the GR gene, which is 19 nucleotides in the cDNA sequence of the gene, is selected.
DNA containing the 27th base site or the 1927th and 2314th base sites is isolated. Isolation of the DNA can be performed, for example, by GR
It can be performed by PCR using chromosomal DNA or RNA as a template, using a primer that hybridizes to the gene. In this method, the isolated DNA is then
The base sequence of is determined. The nucleotide sequence of the isolated DNA can be determined by a method well known to those skilled in the art.

In the present method, the determined DNA is then
The nucleotide sequence of is compared with the control. The control in this method usually refers to the sequence of the normal (wild type) GR gene. Generally, since the sequence of the GR gene of a healthy person is considered to be normal, the term “comparing with the control” in the above step usually means comparing with the sequence of the GR gene of a healthy person. It may be compared with the sequence of the GR gene (GenBank Accession No .: NM 000176) registered as a wild type in.

The test method of the present invention can be carried out by various methods capable of detecting polymorphisms in addition to the method of directly determining the base sequence of DNA derived from a subject as described above.

For example, the polymorphism in the present invention can be detected by the following method. First, a DNA sample is prepared from a subject. Then, the prepared DNA sample is cleaved with a restriction enzyme. Then, the DNA fragments are separated according to their size. Then, the size of the detected DNA fragment is compared with the control. Moreover, in another one embodiment, first, a DNA sample is prepared from a subject. Next, the base site in the GR gene, which is the cD of the gene
A DNA containing the 1927th base site or the 1927th and 2314th base sites in the NA sequence is amplified. Furthermore, the amplified DNA is cut with a restriction enzyme. Then D
NA fragments are separated according to their size. Then, the size of the detected DNA fragment is compared with the control.

As such a method, for example, Restriction Fragment Length Polymorp
hism / RFLP) and PCR-RFLP method. Specifically, if there is a mutation at the recognition site of the restriction enzyme, or if there is a base insertion or deletion in the DNA fragment generated by the restriction enzyme treatment, the size of the fragment generated after the restriction enzyme treatment is compared with that of the control. Change. By amplifying the portion containing this mutation by the PCR method and treating with each restriction enzyme, these mutations can be detected as a difference in the mobility of bands after electrophoresis. Alternatively, the presence or absence of a mutation can be detected by treating chromosomal DNA with these restriction enzymes, performing electrophoresis, and then performing Southern blotting using the probe DNA of the present invention. The restriction enzyme used can be appropriately selected according to each mutation. In this method, in addition to genomic DNA, RNA prepared from a subject can be converted into cDNA with a reverse transcriptase, which can be cleaved as it is with a restriction enzyme and then subjected to Southern blotting. It is also possible to amplify the GR gene-containing DNA by PCR using this cDNA as a template, cut it with a restriction enzyme, and then investigate the difference in mobility.

[0027] In yet another method, first, a DNA sample is prepared from a subject. Then, a DNA containing a base site in the GR gene, which includes the 1927th base site or the 1927th and 2314th base sites in the cDNA sequence of the gene is amplified. In addition, the amplified DNA is converted to single-stranded DN.
Dissociate into A. The dissociated single-stranded DNA is then separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with that of the control.

As the method, for example, PCR-SSCP (single-
strand conformation polymorphism (Cloning and polymerase chain reaction-single
-strand conformation polymorphism analysis of anon
ymous Alu repeats on chromosome 11. Genomics. 1992
Jan 1; 12 (1): 139-146., Detection of p53 gene mut
ations in human brain tumors by single-strand conf
ormation polymorphism analysis of polymerase chain
reaction products. Oncogene. 1991 Aug 1; 6 (8): 131
3-1318., Multiple fluorescence-based PCR-SSCP anal
ysis with postlabeling., PCR Methods Appl. 1995 Ap
r 1; 4 (5): 275-282.). This method is relatively easy to operate and has the advantage that the amount of test sample can be small and so is particularly suitable for screening a large number of DNA samples. The principle is as follows.
When a double-stranded DNA fragment is dissociated into single strands, each strand forms its own higher-order structure depending on its base sequence. When this dissociated DNA chain is electrophoresed in a polyacrylamide gel containing no denaturing agent, complementary single-stranded DNAs of the same chain length move to different positions depending on the difference in their higher-order structures. The single-stranded substitution also changes the higher-order structure of this single-stranded DNA and shows different mobilities in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of a mutation such as a point mutation, a deletion, or an insertion in the DNA fragment.

Specifically, first, the DNA containing the GR gene is
Amplify by CR method. The range of amplification is usually preferably about 200 to 400 bp. Those skilled in the art can perform PCR by appropriately selecting reaction conditions and the like.
At the time of PCR, the amplified DNA product can be labeled by using a primer labeled with an isotope such as ³² P, a fluorescent dye, or biotin. Or PCR
It is also possible to label the amplified DNA product by adding a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin to the reaction solution and performing PCR. Furthermore, in using Klenow enzyme or the like after the PCR reaction, ³² P
Labeling can also be performed by adding a substrate base labeled with an isotope such as, a fluorescent dye, or biotin to the amplified DNA fragment. The labeled DNA fragment thus obtained is denatured by applying heat or the like, and electrophoresed on a polyacrylamide gel containing no denaturing agent such as urea. At this time, the conditions for separating the DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. In addition, although the electrophoresis conditions vary depending on the properties of each DNA fragment, they are usually performed at room temperature (20 to 25 ° C), and when a preferable separation is not obtained, a temperature from 4 to 30 ° C that gives the optimum mobility Consider. After electrophoresis, the mobility of the DNA fragment is detected by autoradiography using an X-ray film, a scanner for detecting fluorescence, or the like, and analyzed. When a band with a difference in mobility is detected, the presence of the mutation can be confirmed by directly cutting out this band from the gel, amplifying it again by PCR, and directly sequencing it. Further, even when the labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide or the silver staining method.

In another method, first, DNA from a subject is
Prepare the sample. Then, a DN which is a base site in the GR gene and includes the 1927th base site or the 1927th and 2314th base sites in the cDNA sequence of the gene.
Amplify A. In addition, the amplified DNA is separated on a gel with increasing concentrations of DNA denaturants. Then separated
The mobility of the DNA on the gel is compared to the control.

As such a method, for example, a denaturant gradient gel electrophore is used.
sis: DGGE method) and the like. The DGGE method is
In a polyacrylamide gel with a denaturant gradient,
In this method, a mixture of DNA fragments is electrophoresed, and the DNA fragments are separated according to the difference in instability. When a labile DNA fragment with a mismatch migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch partially dissociates into single strands due to its instability. The mobility of this partially dissociated DNA fragment is very slow,
Since they differ from the mobility of complete double-stranded DNA with no dissociated portion, they can be separated from each other. Specifically, GR
A DNA containing a gene is amplified by a PCR method using the primer or the like of the present invention, and this is electrophoresed in a polyacrylamide gel that gradually increases as the concentration of a denaturant such as urea shifts. Compare. In the case of a DNA fragment that has a mutation, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the migration speed becomes extremely slow. Therefore, the presence or absence of a mutation can be detected by detecting this mobility difference. be able to.

[0032] Another method is as follows. First, the base site in the GR gene prepared from a subject,
Provided is a DNA containing the 1927th base site or the 1927th and 2314th base sites in the NA sequence, and a substrate on which a nucleotide probe that hybridizes with the DNA is immobilized. Next, the DNA is brought into contact with the substrate. Furthermore, GR gene polymorphism is detected by detecting the DNA hybridized with the nucleotide probe fixed to the substrate.

As such a method, the DNA array method (S
NP gene polymorphism strategy, Kenichi Matsubara / Yoshiyuki Sakaki, Nakayama Shoten, p1
28-135). DN containing GR gene from subject
A sample can be prepared by a method well known to those skilled in the art. In a preferred embodiment of the preparation of the DNA sample, for example, it is prepared on the basis of chromosomal DNA extracted from peripheral blood leukocytes of the subject, skin, tissues or cells such as oral mucous membranes, tears, saliva, urine, feces or hair. You can To prepare a DNA sample of this method from chromosomal DNA, for example, D containing GR gene
Chromosome DN using primers that hybridize to NA
It is also possible to prepare a DNA containing the GR gene by PCR using A as a template. If necessary, the prepared DNA sample can be labeled for detection by a method well known to those skilled in the art.

In the present invention, the "substrate" means a plate-shaped material capable of immobilizing nucleotides. In the present invention, nucleotides include oligonucleotides and polynucleotides. The substrate of the present invention is not particularly limited as long as it can immobilize nucleotides, but substrates generally used in DNA array technology can be preferably used.

Generally, a DNA array is composed of thousands of nucleotides printed on a substrate with high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but porous membranes such as nitrocellulose membranes can be used.

In the present invention, an oligonucleotide-based array developed by Affymetrix can be exemplified as a nucleotide immobilization (array) method. In an array of oligonucleotides, the oligonucleotides are usually synthesized in situ. For example, photoli
thographic technology (Affymetrix) and inkjet for fixing chemicals (Rosetta Inpharmatic
In-situ synthetic methods of oligonucleotides by the technology (s., Inc.) are already known, and any technology can be used for producing the substrate of the present invention.

The nucleotide probe immobilized on the substrate is
If it is possible to detect polymorphism of GR gene,
There is no particular limitation. That is, the probe is a probe that specifically hybridizes with, for example, a wild-type GR gene or a GR gene having a polymorphism. If specific hybridization is possible, the nucleotide probe may be DNA containing the GR gene to be detected or G having a polymorphism.
It need not be completely complementary to the R gene.

In the present invention, the length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bases, preferably 10 to 50, when the oligonucleotide is immobilized.
It is a base, more preferably 15 to 25 bases.

In the present invention, the cDNA sample is then brought into contact with the substrate. By this step, a DNA sample is hybridized with the nucleotide probe. The reaction solution and reaction conditions for hybridization may vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, but generally they can be performed by methods well known to those skilled in the art.

In the present invention, the presence or absence of hybridization or the intensity of the hybridization between the DNA sample and the nucleotide probe immobilized on the substrate is then detected. This detection can be performed, for example, by reading the fluorescence signal with a scanner or the like. In a DNA array, DNA immobilized on a slide glass is generally called a probe, while labeled DNA in a solution is called a target. Therefore, the above nucleotide immobilized on the substrate is referred to as a nucleotide probe in the present specification.

In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used for the purpose of detecting only a mutation at a specific position. When an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is produced and hybridized with this and a sample DNA, the efficiency of hybridization is reduced in the presence of the mutation. It can be detected by Southern blotting or a method utilizing the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. It can also be detected by the ribonuclease A mismatch cleavage method. Specifically, labeled RN prepared by amplifying DNA containing GR gene by PCR etc. and incorporating it into a plasmid vector etc.
Hybridize with A. Since the hybrid has a single-stranded structure in the portion where the mutation exists, the presence of the mutation can be detected by cleaving this portion with ribonuclease A and detecting this by autoradiography or the like.

Other methods capable of detecting the polymorphism of the present invention include (1) a method by mass spectrometry (Griffin TJ a
nd Smith LM, Trends Biotechnol.vol. 18, pp77-84,
(2000)), (2) Method by Taq-Man PCR (Livak KJ. G
enet. Anal. vol.14, pp143-149, (1999)), (3) Pyr
Method by osequencing (Ahmadian A et al., Anal. Bi
ochem. vol. 280, pp103-110, (2000)), (4) Invade
r method (Lyamichev V et al., Nat. Biotechno
l. vol. 17, pp292-296, (1999), Published by Medical Do, "Gene Medicine" vol.4, No.1, pp44-51 and pp68-7
2 (2000)) and the like. Although various detection methods have been exemplified above, the detection method is not limited to these, and enables detection of the polymorphism that reduces the glucocorticoid-binding ability of GR, or the polymorphism that reduces the transcription activation ability of GR. Any method can be used as long as it is a method.

The present invention also provides a polymorphic site in the GR gene which can be used in the test method of the present invention, and which has a chain length of at least 15 nucleotides, including the 1927th polymorphic site in the cDNA sequence of the gene. A polynucleotide having is provided. The base at the polymorphic site is preferably cytosine.

The present invention also provides reagents for testing the efficacy of glucocorticoid formulations. As one embodiment thereof, a DNA that is a base site in the GR gene and contains the 1927th base site in the cDNA sequence of the gene.
It is a reagent for testing the efficacy of a glucocorticoid preparation, which comprises a polynucleotide that hybridizes to and has a chain length of at least 15 nucleotides. The reagent is
Further, it may include a polynucleotide which has a base length in the GR gene and which has a chain length of at least 15 nucleotides and hybridizes to a DNA containing the 2314th base site in the cDNA sequence of the gene.

The oligonucleotide contains a GR gene
It hybridizes specifically to DNA. The term "specifically hybridize" as used herein means ordinary hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laborator).
y Press, New York, USA, 2nd edition 1989) does not significantly cause cross-hybridization with DNAs encoding other proteins. The oligonucleotide need not be completely complementary to the base sequence of the GR gene to be detected, as long as specific hybridization is possible.

The oligonucleotide can be used as a probe or primer in the above-mentioned inspection method of the present invention. When the oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the GR gene including the polymorphic part.

When the above-mentioned oligonucleotide is used as a probe, the probe is specific to a DNA which is a base site in the GR gene and contains the 1927th base site or the 2314th base site in the cDNA sequence of the gene. There is no particular limitation as long as it hybridizes physically. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp or more.

The oligonucleotide of the present invention can be produced, for example, by a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by treatment with a restriction enzyme or the like.

When the oligonucleotide of the present invention is used as a probe, it is preferably labeled appropriately before use. As a method of labeling, using T4 polynucleotide kinase, a method of labeling by phosphorylating the 5'end of an oligonucleotide with ³² P, and a DNA polymerase such as Klenow enzyme, a random hexamer oligonucleotide, etc. As the primer, a method of incorporating a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin (random prime method etc.) can be exemplified.

[0050] Another aspect of the test agent of the present invention is the base site in the GR gene, which is cD of the gene.
It is a reagent for testing the efficacy of a glucocorticoid preparation, which comprises a forward primer designed to amplify a DNA containing the 1927th base site in the NA sequence and a reverse primer. The reagent further comprises GR
It may contain a forward primer and a reverse primer designed to amplify a DNA containing a base site in the gene and a 2314th base site in the cDNA sequence of the gene. The length of the primer is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the GR gene including the polymorphic part.

In the reagent of the present invention, in addition to the active ingredient oligonucleotide, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer,
Protein stabilizers (BSA, gelatin, etc.), preservatives and the like may be mixed if necessary.

[0052]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

(1) DNA source and sequencing of GR gene Ten different Japanese (JCRB0071, 0086, 0091, 009)
2, 0094, 0104, 0105, 0112, 0114 and 0122), and one Japanese colon cancer cell line (JCRB0208) derived from the Health Science Research Resources Bank (HSRR).
B)) (Osaka, Japan) or directly from the National Institute of Health Sciences (NIHS) Ministry of Health, Labor and Welfare Research Resource Bank (Japanes)
e Collection of Research Bioresources (JCRB)) (Tokyo, Japan).

Primer design for the polymerase chain reaction (PCR) and sequencing of the coding region was performed using the sequences (M78506, M78507, U78508, AC00478) obtained from the NCBI database.
Based on 2). All the primers (SEQ ID NOs: 7-30) used in this test are listed in Table 1.

[0055]

[Table 1]

The PCR conditions were as follows: Ex-Taq polymerase (TaKaRa, Kyoto, Japan) is used to
C. for 5 minutes, followed by 95.degree. C. for 30 seconds, 55.degree. C. for 1 minute and 72.degree. C. for 2 minutes for 30 cycles and 72.degree. After that, the PCR product was transferred to the ABI Prism 3700 DNA Analyzer (A
ABI BigDye using pplied Biosystems, CA, USA)
Terminater Cycle Sequencing Kit (Applied Biosyste
Sequencing was performed directly using MS (CA, USA).

(2) Plasmid American Type Culture Collection (ATC
C, VA, USA) pRShGRα contains the full-length coding region of human GRα and the constitutively active Rous sarcoma virus promoter (V. Giguere, SM Hol.
lenberg, MG Rosenfeld, RM Evans, Functional
domains of the human glucocorticoid receptor. Cell
46 (1986) 645-652). Mutant C643R GR and Mutant 231
The expression vector for 4 ins-a GR contains wild type as a template.
The pRShGRα plasmid was used to construct with a QuickChange site-directed mutagenesis kit (Stratagene, CA, USA).
A vector plasmid as a control was constructed by removing the hGRα region from the pRShGRα plasmid.

(3) Cell culture COS-7 cells were obtained from the Research Resource Bank (JCRB) of the Ministry of Health, Labor and Welfare (Tokyo, Japan).
(Japan). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 100 U / ml penicillin and streptomycin in a 5% CO ₂ atmosphere at 37 ° C.

(4) Transiently transfected COS-7 cells were transferred to 10 cm tissue culture dishes (1.6 x 10 ⁶ cells for Western blot and dexamethasone binding assay).
/ Dish) or 6 well tissue culture dishes (1.4 x 10 ⁵ cells / well) for luciferase assay and grown overnight. 4 μg of expression vector encoding either wild-type GR (pRShGRα), C643R GR or 2314 ins-a GR in serum- and antibiotic-free DMEM (per dish for 10 cm dishes) or 1.5 μg (For 6-well dishes, per well) were mixed with PolyFect transfection reagent (Qiagen, Hilden, Germany). This mixture was diluted with 10% FBS-DMEM and immediately added dropwise to the cells. To prepare the cytoplasmic fraction, after transfection and incubation for 48 hours, cells were washed with phosphate buffered saline (PBS) and dispersed by trypsinization. Centrifuge the medium to obtain SET buffer (0.25 M sucrose, 1 mM
After replacing with EDTA, 100 mM Tris-HCl (pH 7.4)), the cells were disrupted by sonication and centrifuged at 105,000 X g.
The protein concentration of the supernatant was measured by BCA protein assay kit (Pierce, IL, USA).

(5) Western blot analysis The cytoplasmic fraction (50 μg protein) was electrophoresed on a 6% SDS-polyacrylamide gel (Tefuco, Tokyo, Japan) and a PVDF membrane (Bio-Rad, CA, Japan) was used. (US). These membranes were stained with a polyclonal rabbit anti-human GR antibody as a primary antibody (Affinity Bioreagents, CO, USA) and a goat anti-rabbit IgG antibody conjugated to alkaline phosphatase as a secondary antibody (Promega, WI, USA). . These signals are transferred to nitroblue tetrazolium chloride (NB
T) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) (Promega, WI, USA) for visualization.

(6) Dexamethasone binding assay A small amount of the cytosolic fraction was [ ³ H] in the presence or absence of a 500-fold excess of unlabeled dexamethasone, respectively.
Dexamethasone (Amersham Pharmacia Biotech, UK)
Incubation with and measured total and non-specific binding, respectively. [ ³ H] dexamethasone binding in the cytosolic fraction was measured using the charcoal adsorption method. Fetal bovine serum albumin (BSA) was added to this solution and the GR
Non-specific adsorption of charcoal on charcoal (Niimi et. A
l., Regulation of glucocorticoid receptor by the t
yrosine kinase inhibitor herbimycin A in the cytos
olic fraction of primary cultured rat hepatocytes.
J. Steroid Biochem. Mol. Biol. 61: 65-71 (1997); N
iimi et.al., Effect of dexamethasone pretreatment
on the dexamethasone-dependent induction of tyros
ine aminotransferase activity in primary cultured
rat hepatocytes. Biol. Pharm. Bull. 21: 1009-1012
(1998)). After centrifugation, the radioactivity of the supernatant was measured by ACSII in a scintillation spectrometer.

(7) Luciferase activity COS-7 cells were transformed with wild-type GR expression vector pRShGRα and mutant G
R expression vector pRShGRα-C643R or pRShGRα-23
The mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc) (ATCC, V
A, USA). 30
After the hour incubation, dexamethasone was added to these cells, which were then incubated for another 18 hours. These cells were washed with PBS, and the lysate was added to the Pica Gene Dual SeaPansy Luminescence Kit (Nip
pongene, Tokyo, Japan) and then luciferase activity was measured using ARVO luminometer (Wallac, Turk).
u, Finland). phRL-TK vector
(Promega, WI, USA) was used as an internal control.

[Example 1] Mutation in exon 7 of GR gene C643R and mutation in exon 9α 2314 ins-a The present inventors sequenced the GR gene coding region of 10 Japanese leukemia cells. Reported to affect susceptibility to glucocorticoids in leukemic CEM-1 cells (S. Geley, BL Hartmann, M. Hala,
EM Strasser-Wozak, K. Kapelari, R. Kofler, Resi
stance to glucocorticoid-induced apoptosis in huma
n T-cell acute lymphoblastic leukemia CEM-C1 cells
is dueto insufficient glucocorticoid receptor exp
No L753F mutation was identified in ression. Cancer Res. 56 (1996) 5033-5038). But the reference array
(NCBI accession number; AC004782) (surrounding sequence
ence); tacgaccaaTgtaaacacat (SEQ ID NO: 31)), and mutation t5618c (C643) in which cysteine-643 is replaced with arginine in exon 7 of the GR gene.
R) was detected. This was a mutation in which the 5618th thymine was replaced with cytosine (in the GR gene cDNA sequence (SEQ ID NO: 1), the 1927th thymine was replaced with cytosine). This mutation was detected in P30 / OHK cell line (HSRRB accession number: JCRBOO94), which is peripheral blood obtained from Japanese acute lymphoblastic leukemia (M. Hanada, M. Sh.
imoyama, Potential limitation of growth-inhibitory
action of recombinant human tumor necrosis factor
(PAC-4D) due to easy induction of resistance: evi
dence in vitro. Jpn. J. Cancer Res. 78 (1987) 1266
-1273; M. Hirose, K. Minato, K. Tobinai, M. Ohira,
T. Ise, S. Watanabe, M. Shimoyama, M. Taniwaki, T.
Abe, A novel pre-T cell line derived from acute l
ymphoblastic leukemia. Gann 73 (1982) 600-605; M.
Hirose, K. Tobinai, K. Minato, M. Ohira, T. Ise, S.
Watanabe, M. Shimoyama, M. Taniwaki, T. Abe, A no
vel cultured cell line (P30 / Ohkubo) of pre-T cell
(C / T hybrid) phenotype derived from an acute lymph
oblastic leukemia with some phenotypic change duri
ng clinical course.Nippon Ketsueki Gakkai Zasshi
46 (1983) 729-736). Japanese colon cancer cell line
In CCK-81 (HSRRB accession number; JCRBO208), mutation 2314 ins-a (cD of GR gene, which replaces leucine-772 to lysine-777 with another 26 amino acids, on exon 9α of GR gene
2314 of the NA sequence (SEQ ID NO: 1) and 2314
A mutation in which adenine was inserted between the 2nd base) was detected.

Next, wild-type GR plasmid, C643R mutant GR plasmid or 2314 ins-a mutant G was added to COS-7 cells.
The R plasmid was transfected, and Western blot analysis of the cytoplasmic fraction was performed using anti-GR antibody. As a result, it was shown that both wild type GR and C643R mutant GR in transfected COS-7 cells were expressed at similar levels (FIG. 1). On the other hand, 2314 ins-in the cytoplasmic fraction of transfected COS-7 cells.
The expression level of a mutant GR was found to be lower than that of wild type GR (Fig. 2). However, in the whole cell lysate, no decrease in the expression level was observed even with 2314 ins-a mutant GR (Fig. 3). From these results, it was shown that the C643R mutation and the 2314 ins-a mutation do not change the expression of GR, but the 2314 ins-a mutation changes the subcellular localization of the expression. In addition, C643R mutant GR and 2
It was shown that the stability of 314 ins-a mutant GR was similar to that of wild type GR.

Example 2 C643R mutant GR and 2314 in
Loss of Transcriptional Activity in sa Mutant GR We used C643R mutant GR and 2314 ins-a mutant G
To examine the transcription activity of R, wild-type GR plasmid, C643R mutant GR plasmid or 2314 ins-a mutant G
The R plasmid was used to transform the mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc) (Ge
nBank; deposit number AF093686) was used to transfect COS-7 cells. The MMTV promoter is widely used as a model system to study glucocorticoid-mediated gene regulation mechanisms (RD Med
h, TJ Schmidt, Trans-retinoic acid and glucocor
ticoids synergistically induce transcription from
the mouse mammary tumorvirus promoter in human emb
ryonic kidney cells. J. Steroid Biochem. Mol. Biol.
62 (1997) 129-142) and contains a well-characterized GR binding sequence (GRE) that mediates hormone induction. The result of the reporter gene experiment is shown in FIG.

Transfection of COS-7 cells with the reporter plasmid alone did not show an increase in luciferase activity in response to dexamethasone, indicating that these cells do not contain a significant amount of functional GR. Luciferase activity increased in a dose-dependent manner in cells transfected with wild-type GR. However, the C643R mutant GR did not induce the expression of the dexamethasone-responsive luciferase reporter gene (Fig. 4). Similar results were obtained with the 2314 ins-a mutant GR. These results show that C643R mutant GR and 2314 in
It was shown that the transcriptional activity of sa mutant GR was suppressed.

Example 3 Reduction of Dexamethasone Binding Ability (Binding Affinity) of C643R Mutant GR Cysteine-643 residue is the ligand binding domain of GR (LB
Located in D). To examine the affinity of C643R mutant GR for dexamethasone, we used Scatchar
The binding affinity to [ ³ H] dexamethasone in the cytosolic fraction of COS-7 cells transfected with wild-type or C643R mutant GR plasmid was measured according to d-plot analysis (G. Scatchard, The aggregation of proteins f.
or small molecules, ions. Ann. NY Acad. Sci. 51
(1949) 660-672). The results of the radioligand binding assay are shown in FIG. 5, and at the same time, the Kd value and the binding ability are shown in Table 2. Specific high affinity binding was revealed in cytosolic fractions prepared from COS-7 cells transfected with wild type GR. This binding capacity was 864 fmol / mg cytoplasmic protein, and the dissociation constant Kd was 7.4 nM. C643R variant
The binding capacity (921 fmol / mg cytoplasmic protein) of the cytosolic fraction from GR-transfected COS-7 cells was comparable to that of the wild type, but the Kd value (44.6nM) was 6 times higher.
From this result, it was shown that the binding affinity of C643R mutant GR to dexamethasone was low.

[0068]

[Table 2]

Glucocorticoid resistance is a major problem in the treatment of acute lymphocytic leukemia, and although some reports have been published on the underlying mechanisms, very little is known. The mutation L753F has been reported to be detected in human steroid resistant leukemia CEM-C1 cells and ICR27TK.3 cells, and the correlation between this mutation and resistance has been discussed (S. Gele
y, BL Hartmann, M. Hala, EM Strasser-Wozak,
K. Kapelari, R. Kofler, Resistance to glucocortico
id-induced apoptosis in human T-cell acute lymphob
lastic leukemia CEM-C1 cells is due to insufficient
glucocorticoid receptor expression.Cancer Res. 56
(1996) 5033-5038).

The present inventors sequenced exon 9α of the GR gene in 10 individual leukemia cells of Japanese to investigate whether the mutation L753F was present in other leukemia cells. Mutant L753F was not detected in the cells. We have found that another mutation is the GR in these cells.
Assumed to be present in the gene, the other coding regions of the GR gene were therefore sequenced. Among them, the present inventors
Another mutation C643R in the ligand binding domain (LBD) and hsp90 binding site in GR gene exon 7 of P30 / OHK cells
Was identified. The P30 / OHK cells have been established from Japanese myeloid leukemia cells suffering from acute lymphoblastic leukemia (M. Hirose, K. Minato, K. Tobinai, M. Ohira,
T. Ise, S. Watanabe, M. Shimoyama, M. Taniwaki,
T. Abe, Anovel pre-T cell line derived from acute
lymphoblastic leukemia. Gann 73 (1982) 600-605; M.
Hirose, K. Tobinai, K. Minato, M. Ohira, T. Ise,
S.Watanabe, M. Shimoyama, M. Taniwaki, T. Abe, A n
ovel cultured cell line (P30 / Ohkubo) of pre-T cell
(C / T hybrid) phenotype derived from an acutelympho
blastic leukemia with some phenotypic change durin
g clinical course. Nippon Ketsueki Gakkai Zasshi 4
6 (1983) 729-736). To investigate the effect of C643R mutant GR, ligand binding affinity and transcriptional activity were tested. As a result, it was shown that C643R mutant GR has a dexamethasone binding affinity 1/6 that of wild-type GR (Fig. 5, Table 2) and little or no transcriptional activity (Fig. 4). It was

Cysteine has been reported to play an important function by promoting the interaction of specific ligands in human, rat and mouse GR.
Human GR LBD has 5 cysteine residues at positions 622 and 638.
Nos. 643, 665 and 736, and these 5 cysteines are conserved in human, rat and mouse GR. Furthermore, Cys-622, -63 of human GR, respectively.
Cys-640, -656 of rat GR homologous to 8, -643, and
-661 is GR's flexible steroid binding cavity
And Cys-643 is involved in the binding pocket (binding
et) near the entrance (PK Chakraborti,
ML Garabedian, KR Yamamoto, SS Simons Jr.,
Role of cysteines 640, 656, and 661 in steroid bi
nding to rat glucocorticoid receptors. J. Biol. Ch
em. 267 (1992) 11366-11373; SS Simons Jr., W.
B. Pratt, Glucocorticoid receptor thiols and stero
id-binding activity.Methods Enzymol.251 (1995) 4
06-422). Of these, the substitution of cysteine 643 for serine essentially retained the wild-type affinity and bioactivity, suggesting that the C643 thiol is not important (C. Yu, N. Warriar, MV Govindan, Cy
steines 638 and 665 in the hormone binding domain
of human glucocorticoid receptor define the specif
icity to glucocorticoids. Biochemistry 34 (1995) 1
4163-14173). However, in this example, substitution of cysteine 643 for arginine resulted in low glucocorticoid binding affinity. These results are Ar
It is suggested that g-643, by its long side chain, interferes with the binding between the steroid binding pocket of GR and dexamethasone. Therefore, the present inventors considered that the change of cysteine 643 to arginine would cause a change in the configuration of the glucocorticoid-binding sinus and reduce the function of binding to glucocorticoid.

GR is two protein isoforms GRα
And GRβ, which result from alternative splicing of the primary transcript of the GR mRNA precursor. GRβ is
It does not bind to glucocorticoids and is an inhibitor of GRα activity. Recently, Strickland et al. (Strickland et.
l., High constitutive glucocorticoid receptor beta
in human neutrophils enables them to reduce their
spontaneous rate of cell death in response to cort
icosteroids. J Exp Med. 193: 585-94. (2001)) showed that constitutive expression of the splicing mutant GRβ reduces cell death in human neutrophils. In P30 / OHK cells, wild type and C643R mutant GR genes are heterozygous. We found that the C643R mutant GR acts like the alternative splice mutant GRβ, resulting in C6.
It was shown that the 43R mutant GR not only has less transcriptional activity than itself, but also acts as an inhibitor of GRα activity. Therefore, the present inventors have found that the mutation C643R
Influences the response to glucocorticoids, and
It suggests that apoptosis can be suppressed in P30 / OHK cells.

The present inventors have found that in the coding regions of 9 other leukemia cells, non-synonyms of amino acids are used.
s) We did not detect any mutations that would cause a change. In these 9 cells, several other possibilities were considered. One possibility is that certain mutations affecting GR mRNA expression may be present at GR transcriptional regulatory sites. Yet another possibility is that certain mutations in the intron region of the GR gene may give rise to splice variants of GR. Therefore, in order to examine these possibilities, the sequences of the transcriptional regulatory site and intron region of GR gene in these 9 types of cells were considered to be interesting.

The above findings indicate that there is a mutation C643R in exon 7 of the GR gene of human leukemic P30 / OHK cells, and that this mutant C643R has less glucocorticoid binding affinity and transcriptional activity. There is. Further, it is shown that the amino acid residue 643 is important in the glucocorticoid-binding ability of GR, and that the mutant C643R affects the transcriptional activity and induces glucocorticoid resistance in P30 / OHK cells.

On the other hand, the present inventors have shown that 2314 ins-a mutant GR has a change in intracellular localization and a loss of transcription activation ability. This gene mutation has already been found in 8 out of 39 Chinese patients with lupus nephritis (Jiang T et al., Thephase-shift mutation in t
he glucocorticoid receptor gene: potential etiolog
ic significance of neuroendocrine mechanisms in lu
pus nephritis. ClinChim Acta. 313: 113-117 (200
1)). In this report, the amount of dexamethasone binding was decreased in 8 patients with mutations, but the amount of dexamethasone binding was also decreased in patients without this mutation. The correlation with was unknown. The present inventors have shown that this 2314 ins-a mutant GR loses transcriptional ability, and found for the first time that this mutation results in loss of GR function (FIG. 4).

There are two nuclear localization signals on the GR protein, which are mutated by this 2314 ins-a mutation.
The 72nd to 777th amino acids correspond to the second nuclear localization signal portion. GR is normally localized in the cytoplasm and translocates from the cytoplasm to the nucleus upon binding of the ligand glucocorticoid to exert transcriptional activity.However, in the 2314 ins-a mutation, GR is localized in the nucleus even if glucocorticoid is not bound. It is possible that it has moved. GR for whole cell lysates
The expression level was not changed between wild type GR and 2314 ins-a mutant GR (Fig. 3), but 2314 ins-a mutant G was observed in the cytoplasm.
The result that the expression level of R is decreased (Fig. 2) is
It supports this idea.

[0077]

EFFECTS OF THE INVENTION The present inventors have found a relationship between GR gene polymorphisms that reduce the glucocorticoid-binding ability of GR or the transcriptional activation ability of GR and the efficacy of glucocorticoid preparations. Was issued. From this,
INDUSTRIAL APPLICABILITY The present invention provides a method for examining the effectiveness of a glucocorticoid preparation in a subject and a test reagent that can be used in the method. The present invention is highly expected to be able to predict the efficacy of a glucocorticoid preparation and to enable the development of an appropriate treatment method for patients who are resistant to the glucocorticoid preparation.

[0078]

[Sequence list]                                SEQUENCE LISTING        <110> National Institute of Health Sciences       The Organization for Pharmaceutical Safety and Research       Genox Research, Inc. <120> Methods for evaluating the effectiveness of glucocorticoid on a pa tient <130> G1-A0116 <140> <141> <160> 31 <170> PatentIn Ver. 2.1 <210> 1 <211> 2334 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (2331) <400> 1 atg gac tcc aaa gaa tca tta act cct ggt aga gaa gaa aac ccc agc 48 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser   1 5 10 15 agt gtg ctt gct cag gag agg gga gat gtg atg gac ttc tat aaa acc 96 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr              20 25 30 cta aga gga gga gct act gtg aag gtt tct gcg tct tca ccc tca ctg 144 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu          35 40 45 gct gtc gct tct caa tca gac tcc aag cag cga aga ctt ttg gtt gat 192 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp      50 55 60 ttt cca aaa ggc tca gta agc aat gcg cag cag cca gat ctg tcc aaa 240 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys  65 70 75 80 gca gtt tca ctc tca atg gga ctg tat atg gga gag aca gaa aca aaa 288 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys                  85 90 95 gtg atg gga aat gac ctg gga ttc cca cag cag ggc caa atc agc ctt 336 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu             100 105 110 tcc tcg ggg gaa aca gac tta aag ctt ttg gaa gaa agc att gca aac 384 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn         115 120 125 ctc aat agg tcg acc agt gtt cca gag aac ccc aag agt tca gca tcc 432 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser     130 135 140 act gct gtg tct gct gcc ccc aca gag aag gag ttt cca aaa act cac 480 Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 tct gat gta tct tca gaa cag caa cat ttg aag ggc cag act ggc acc 528 Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr                 165 170 175 aac ggt ggc aat gtg aaa ttg tat acc aca gac caa agc acc ttt gac 576 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp             180 185 190 att ttg cag gat ttg gag ttt tct tct ggg tcc cca ggt aaa gag acg 624 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr         195 200 205 aat gag agt cct tgg aga tca gac ctg ttg ata gat gaa aac tgt ttg 672 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu     210 215 220 ctt tct cct ctg gcg gga gaa gac gat tca ttc ctt ttg gaa gga aac 720 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 tcg aat gag gac tgc aag cct ctc att tta ccg gac act aaa ccc aaa 768 Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys                 245 250 255 att aag gat aat gga gat ctg gtt ttg tca agc ccc agt aat gta aca 816 Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr             260 265 270 ctg ccc caa gtg aaa aca gaa aaa gaa gat ttc atc gaa ctc tgc acc 864 Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr         275 280 285 cct ggg gta att aag caa gag aaa ctg ggc aca gtt tac tgt cag gca 912 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala     290 295 300 agc ttt cct gga gca aat ata att ggt aat aaa atg tct gcc att tct 960 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320 gtt cat ggt gtg agt acc tct gga gga cag atg tac cac tat gac atg 1008 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met                 325 330 335 aat aca gca tcc ctt tct caa cag cag gat cag aag cct att ttt aat 1056 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn             340 345 350 gtc att cca cca att ccc gtt ggt tcc gaa aat tgg aat agg tgc caa 1104 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln         355 360 365 gga tct gga gat gac aac ttg act tct ctg ggg act ctg aac ttc cct 1152 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro     370 375 380 ggt cga aca gtt ttt tct aat ggc tat tca agc ccc agc atg aga cca 1200 Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 gat gta agc tct cct cca tcc agc tcc tca aca gca aca aca gga cca 1248 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro                 405 410 415 cct ccc aaa ctc tgc ctg gtg tgc tct gat gaa gct tca gga tgt cat 1296 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His             420 425 430 tat gga gtc tta act tgt gga agc tgt aaa gtt ttc ttc aaa aga gca 1344 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala         435 440 445 gtg gaa gga cag cac aat tac cta tgt gct gga agg aat gat tgc atc 1392 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile     450 455 460 atc gat aaa att cga aga aaa aac tgc cca gca tgc cgc tat cga aaa 1440 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 tgt ctt cag gct gga atg aac ctg gaa gct cga aaa aca aag aaa aaa 1488 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys                 485 490 495 ata aaa gga att cag cag gcc act aca gga gtc tca caa gaa acc tct 1536 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser             500 505 510 gaa aat cct ggt aac aaa aca ata gtt cct gca acg tta cca caa ctc 1584 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu         515 520 525 acc cct acc ctg gtg tca ctg ttg gag gtt att gaa cct gaa gtg tta 1632 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu     530 535 540 tat gca gga tat gat agc tct gtt cca gac tca act tgg agg atc atg 1680 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545 550 555 560 act acg ctc aac atg tta gga ggg cgg caa gtg att gca gca gtg aaa 1728 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys                 565 570 575 tgg gca aag gca ata cca ggt ttc agg aac tta cac ctg gat gac caa 1776 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln             580 585 590 atg acc cta ctg cag tac tcc tgg atg ttt ctt atg gca ttt gct ctg 1824 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu         595 600 605 ggg tgg aga tca tat aga caa tca agt gca aac ctg ctg tgt ttt gct 1872 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala     610 615 620 cct gat ctg att att aat gag cag aga atg act cta ccc tgc atg tac 1920 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 gac caa tgt aaa cac atg ctg tat gtt tcc tct gag tta cac agg ctt 1968 Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu                 645 650 655 cag gta tct tat gaa gag tat ctc tgt atg aaa acc tta ctg ctt ctc 2016 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu             660 665 670 tct tca gtt cct aag gac ggt ctg aag agc caa gag cta ttt gat gaa 2064 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu         675 680 685 att aga atg acc tac atc aaa gag cta gga aaa gcc att gtc aag agg 2112 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg     690 695 700 gaa gga aac tcc agc cag aac tgg cag cgg ttt tat caa ctg aca aaa 2160 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 ctc ttg gat tct atg cat gaa gtg gtt gaa aat ctc ctt aac tat tgc 2208 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys                 725 730 735 ttc caa aca ttt ttg gat aag acc atg agt att gaa ttc ccc gag atg 2256 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met             740 745 750 tta gct gaa atc atc acc aat cag ata cca aaa tat tca aat gga aat 2304 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn         755 760 765 atc aaa aaa ctt ctg ttt cat caa aag tga 2334 Ile Lys Lys Leu Leu Phe His Gln Lys     770 775 <210> 2 <211> 777 <212> PRT <213> Homo sapiens <400> 2 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser   1 5 10 15 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr              20 25 30 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu          35 40 45 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp      50 55 60 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys  65 70 75 80 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys                  85 90 95 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu             100 105 110 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn         115 120 125 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser     130 135 140 Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr                 165 170 175 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp             180 185 190 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr         195 200 205 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu     210 215 220 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys                 245 250 255 Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr             260 265 270 Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr         275 280 285 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala     290 295 300 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met                 325 330 335 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn             340 345 350 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln         355 360 365 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro     370 375 380 Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro                 405 410 415 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His             420 425 430 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala         435 440 445 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile     450 455 460 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys                 485 490 495 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser             500 505 510 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu         515 520 525 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu     530 535 540 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545 550 555 560 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys                 565 570 575 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln             580 585 590 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu         595 600 605 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala     610 615 620 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu                 645 650 655 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu             660 665 670 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu         675 680 685 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg     690 695 700 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys                 725 730 735 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met             740 745 750 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn         755 760 765 Ile Lys Lys Leu Leu Phe His Gln Lys     770 775 <210> 3 <211> 2334 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (2331) <400> 3 atg gac tcc aaa gaa tca tta act cct ggt aga gaa gaa aac ccc agc 48 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser   1 5 10 15 agt gtg ctt gct cag gag agg gga gat gtg atg gac ttc tat aaa acc 96 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr              20 25 30 cta aga gga gga gct act gtg aag gtt tct gcg tct tca ccc tca ctg 144 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu          35 40 45 gct gtc gct tct caa tca gac tcc aag cag cga aga ctt ttg gtt gat 192 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp      50 55 60 ttt cca aaa ggc tca gta agc aat gcg cag cag cca gat ctg tcc aaa 240 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys  65 70 75 80 gca gtt tca ctc tca atg gga ctg tat atg gga gag aca gaa aca aaa 288 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys                  85 90 95 gtg atg gga aat gac ctg gga ttc cca cag cag ggc caa atc agc ctt 336 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu             100 105 110 tcc tcg ggg gaa aca gac tta aag ctt ttg gaa gaa agc att gca aac 384 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn         115 120 125 ctc aat agg tcg acc agt gtt cca gag aac ccc aag agt tca gca tcc 432 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser     130 135 140 act gct gtg tct gct gcc ccc aca gag aag gag ttt cca aaa act cac 480 Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 tct gat gta tct tca gaa cag caa cat ttg aag ggc cag act ggc acc 528 Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr                 165 170 175 aac ggt ggc aat gtg aaa ttg tat acc aca gac caa agc acc ttt gac 576 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp             180 185 190 att ttg cag gat ttg gag ttt tct tct ggg tcc cca ggt aaa gag acg 624 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr         195 200 205 aat gag agt cct tgg aga tca gac ctg ttg ata gat gaa aac tgt ttg 672 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu     210 215 220 ctt tct cct ctg gcg gga gaa gac gat tca ttc ctt ttg gaa gga aac 720 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 tcg aat gag gac tgc aag cct ctc att tta ccg gac act aaa ccc aaa 768 Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys                 245 250 255 att aag gat aat gga gat ctg gtt ttg tca agc ccc agt aat gta aca 816 Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr             260 265 270 ctg ccc caa gtg aaa aca gaa aaa gaa gat ttc atc gaa ctc tgc acc 864 Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr         275 280 285 cct ggg gta att aag caa gag aaa ctg ggc aca gtt tac tgt cag gca 912 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala     290 295 300 agc ttt cct gga gca aat ata att ggt aat aaa atg tct gcc att tct 960 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320 gtt cat ggt gtg agt acc tct gga gga cag atg tac cac tat gac atg 1008 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met                 325 330 335 aat aca gca tcc ctt tct caa cag cag gat cag aag cct att ttt aat 1056 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn             340 345 350 gtc att cca cca att ccc gtt ggt tcc gaa aat tgg aat agg tgc caa 1104 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln         355 360 365 gga tct gga gat gac aac ttg act tct ctg ggg act ctg aac ttc cct 1152 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro     370 375 380 ggt cga aca gtt ttt tct aat ggc tat tca agc ccc agc atg aga cca 1200 Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 gat gta agc tct cct cca tcc agc tcc tca aca gca aca aca gga cca 1248 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro                 405 410 415 cct ccc aaa ctc tgc ctg gtg tgc tct gat gaa gct tca gga tgt cat 1296 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His             420 425 430 tat gga gtc tta act tgt gga agc tgt aaa gtt ttc ttc aaa aga gca 1344 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala         435 440 445 gtg gaa gga cag cac aat tac cta tgt gct gga agg aat gat tgc atc 1392 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile     450 455 460 atc gat aaa att cga aga aaa aac tgc cca gca tgc cgc tat cga aaa 1440 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 tgt ctt cag gct gga atg aac ctg gaa gct cga aaa aca aag aaa aaa 1488 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys                 485 490 495 ata aaa gga att cag cag gcc act aca gga gtc tca caa gaa acc tct 1536 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser             500 505 510 gaa aat cct ggt aac aaa aca ata gtt cct gca acg tta cca caa ctc 1584 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu         515 520 525 acc cct acc ctg gtg tca ctg ttg gag gtt att gaa cct gaa gtg tta 1632 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu     530 535 540 tat gca gga tat gat agc tct gtt cca gac tca act tgg agg atc atg 1680 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545 550 555 560 act acg ctc aac atg tta gga ggg cgg caa gtg att gca gca gtg aaa 1728 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys                 565 570 575 tgg gca aag gca ata cca ggt ttc agg aac tta cac ctg gat gac caa 1776 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln             580 585 590 atg acc cta ctg cag tac tcc tgg atg ttt ctt atg gca ttt gct ctg 1824 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu         595 600 605 ggg tgg aga tca tat aga caa tca agt gca aac ctg ctg tgt ttt gct 1872 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala     610 615 620 cct gat ctg att att aat gag cag aga atg act cta ccc tgc atg tac 1920 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 gac caa cgt aaa cac atg ctg tat gtt tcc tct gag tta cac agg ctt 1968 Asp Gln Arg Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu                 645 650 655 cag gta tct tat gaa gag tat ctc tgt atg aaa acc tta ctg ctt ctc 2016 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu             660 665 670 tct tca gtt cct aag gac ggt ctg aag agc caa gag cta ttt gat gaa 2064 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu         675 680 685 att aga atg acc tac atc aaa gag cta gga aaa gcc att gtc aag agg 2112 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg     690 695 700 gaa gga aac tcc agc cag aac tgg cag cgg ttt tat caa ctg aca aaa 2160 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 ctc ttg gat tct atg cat gaa gtg gtt gaa aat ctc ctt aac tat tgc 2208 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys                 725 730 735 ttc caa aca ttt ttg gat aag acc atg agt att gaa ttc ccc gag atg 2256 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met             740 745 750 tta gct gaa atc atc acc aat cag ata cca aaa tat tca aat gga aat 2304 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn         755 760 765 atc aaa aaa ctt ctg ttt cat caa aag tga 2334 Ile Lys Lys Leu Leu Phe His Gln Lys     770 775 <210> 4 <211> 777 <212> PRT <213> Homo sapiens <400> 4 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser   1 5 10 15 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr              20 25 30 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu          35 40 45 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp      50 55 60 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys  65 70 75 80 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys                  85 90 95 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu             100 105 110 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn         115 120 125 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser     130 135 140 Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr                 165 170 175 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp             180 185 190 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr         195 200 205 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu     210 215 220 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys                 245 250 255 Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr             260 265 270 Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr         275 280 285 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala     290 295 300 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met                 325 330 335 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn             340 345 350 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln         355 360 365 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro     370 375 380 Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro                 405 410 415 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His             420 425 430 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala         435 440 445 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile     450 455 460 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys                 485 490 495 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser             500 505 510 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu         515 520 525 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu     530 535 540 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545 550 555 560 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys                 565 570 575 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln             580 585 590 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu         595 600 605 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala     610 615 620 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 Asp Gln Arg Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu                 645 650 655 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu             660 665 670 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu         675 680 685 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg     690 695 700 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys                 725 730 735 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met             740 745 750 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn         755 760 765 Ile Lys Lys Leu Leu Phe His Gln Lys     770 775 <210> 5 <211> 2394 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (2391) <400> 5 atg gac tcc aaa gaa tca tta act cct ggt aga gaa gaa aac ccc agc 48 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser   1 5 10 15 agt gtg ctt gct cag gag agg gga gat gtg atg gac ttc tat aaa acc 96 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr              20 25 30 cta aga gga gga gct act gtg aag gtt tct gcg tct tca ccc tca ctg 144 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu          35 40 45 gct gtc gct tct caa tca gac tcc aag cag cga aga ctt ttg gtt gat 192 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp      50 55 60 ttt cca aaa ggc tca gta agc aat gcg cag cag cca gat ctg tcc aaa 240 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys  65 70 75 80 gca gtt tca ctc tca atg gga ctg tat atg gga gag aca gaa aca aaa 288 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys                  85 90 95 gtg atg gga aat gac ctg gga ttc cca cag cag ggc caa atc agc ctt 336 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu             100 105 110 tcc tcg ggg gaa aca gac tta aag ctt ttg gaa gaa agc att gca aac 384 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn         115 120 125 ctc aat agg tcg acc agt gtt cca gag aac ccc aag agt tca gca tcc 432 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser     130 135 140 act gct gtg tct gct gcc ccc aca gag aag gag ttt cca aaa act cac 480 Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 tct gat gta tct tca gaa cag caa cat ttg aag ggc cag act ggc acc 528 Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr                 165 170 175 aac ggt ggc aat gtg aaa ttg tat acc aca gac caa agc acc ttt gac 576 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp             180 185 190 att ttg cag gat ttg gag ttt tct tct ggg tcc cca ggt aaa gag acg 624 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr         195 200 205 aat gag agt cct tgg aga tca gac ctg ttg ata gat gaa aac tgt ttg 672 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu     210 215 220 ctt tct cct ctg gcg gga gaa gac gat tca ttc ctt ttg gaa gga aac 720 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 tcg aat gag gac tgc aag cct ctc att tta ccg gac act aaa ccc aaa 768 Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys                 245 250 255 att aag gat aat gga gat ctg gtt ttg tca agc ccc agt aat gta aca 816 Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr             260 265 270 ctg ccc caa gtg aaa aca gaa aaa gaa gat ttc atc gaa ctc tgc acc 864 Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr         275 280 285 cct ggg gta att aag caa gag aaa ctg ggc aca gtt tac tgt cag gca 912 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala     290 295 300 agc ttt cct gga gca aat ata att ggt aat aaa atg tct gcc att tct 960 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320 gtt cat ggt gtg agt acc tct gga gga cag atg tac cac tat gac atg 1008 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met                 325 330 335 aat aca gca tcc ctt tct caa cag cag gat cag aag cct att ttt aat 1056 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn             340 345 350 gtc att cca cca att ccc gtt ggt tcc gaa aat tgg aat agg tgc caa 1104 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln         355 360 365 gga tct gga gat gac aac ttg act tct ctg ggg act ctg aac ttc cct 1152 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro     370 375 380 ggt cga aca gtt ttt tct aat ggc tat tca agc ccc agc atg aga cca 1200 Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 gat gta agc tct cct cca tcc agc tcc tca aca gca aca aca gga cca 1248 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro                 405 410 415 cct ccc aaa ctc tgc ctg gtg tgc tct gat gaa gct tca gga tgt cat 1296 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His             420 425 430 tat gga gtc tta act tgt gga agc tgt aaa gtt ttc ttc aaa aga gca 1344 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala         435 440 445 gtg gaa gga cag cac aat tac cta tgt gct gga agg aat gat tgc atc 1392 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile     450 455 460 atc gat aaa att cga aga aaa aac tgc cca gca tgc cgc tat cga aaa 1440 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 tgt ctt cag gct gga atg aac ctg gaa gct cga aaa aca aag aaa aaa 1488 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys                 485 490 495 ata aaa gga att cag cag gcc act aca gga gtc tca caa gaa acc tct 1536 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser             500 505 510 gaa aat cct ggt aac aaa aca ata gtt cct gca acg tta cca caa ctc 1584 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu         515 520 525 acc cct acc ctg gtg tca ctg ttg gag gtt att gaa cct gaa gtg tta 1632 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu     530 535 540 tat gca gga tat gat agc tct gtt cca gac tca act tgg agg atc atg 1680 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545 550 555 560 act acg ctc aac atg tta gga ggg cgg caa gtg att gca gca gtg aaa 1728 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys                 565 570 575 tgg gca aag gca ata cca ggt ttc agg aac tta cac ctg gat gac caa 1776 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln             580 585 590 atg acc cta ctg cag tac tcc tgg atg ttt ctt atg gca ttt gct ctg 1824 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu         595 600 605 ggg tgg aga tca tat aga caa tca agt gca aac ctg ctg tgt ttt gct 1872 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala     610 615 620 cct gat ctg att att aat gag cag aga atg act cta ccc tgc atg tac 1920 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 gac caa tgt aaa cac atg ctg tat gtt tcc tct gag tta cac agg ctt 1968 Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu                 645 650 655 cag gta tct tat gaa gag tat ctc tgt atg aaa acc tta ctg ctt ctc 2016 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu             660 665 670 tct tca gtt cct aag gac ggt ctg aag agc caa gag cta ttt gat gaa 2064 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu         675 680 685 att aga atg acc tac atc aaa gag cta gga aaa gcc att gtc aag agg 2112 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg     690 695 700 gaa gga aac tcc agc cag aac tgg cag cgg ttt tat caa ctg aca aaa 2160 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 ctc ttg gat tct atg cat gaa gtg gtt gaa aat ctc ctt aac tat tgc 2208 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys                 725 730 735 ttc caa aca ttt ttg gat aag acc atg agt att gaa ttc ccc gag atg 2256 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met             740 745 750 tta gct gaa atc atc acc aat cag ata cca aaa tat tca aat gga aat 2304 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn         755 760 765 atc aaa aaa act tct gtt tca tca aaa gtg act gcc tta ata aga atg 2352 Ile Lys Lys Thr Ser Val Ser Ser Lys Val Thr Ala Leu Ile Arg Met     770 775 780 gtt gcc tta aag aaa gtc gaa tta ata gct ttt att gta taa 2394 Val Ala Leu Lys Lys Val Glu Leu Ile Ala Phe Ile Val 785 790 795 <210> 6 <211> 797 <212> PRT <213> Homo sapiens <400> 6 Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser   1 5 10 15 Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr              20 25 30 Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu          35 40 45 Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp      50 55 60 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys  65 70 75 80 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys                  85 90 95 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu             100 105 110 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn         115 120 125 Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser     130 135 140 Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr                 165 170 175 Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp             180 185 190 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr         195 200 205 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu     210 215 220 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn 225 230 235 240 Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys                 245 250 255 Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr             260 265 270 Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr         275 280 285 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala     290 295 300 Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310 315 320 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met                 325 330 335 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn             340 345 350 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln         355 360 365 Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro     370 375 380 Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro                 405 410 415 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His             420 425 430 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala         435 440 445 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile     450 455 460 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 465 470 475 480 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys                 485 490 495 Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser             500 505 510 Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu         515 520 525 Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu     530 535 540 Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met 545 550 555 560 Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys                 565 570 575 Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln             580 585 590 Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu         595 600 605 Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala     610 615 620 Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr 625 630 635 640 Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu                 645 650 655 Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu             660 665 670 Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu         675 680 685 Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg     690 695 700 Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys 705 710 715 720 Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys                 725 730 735 Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met             740 745 750 Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn         755 760 765 Ile Lys Lys Thr Ser Val Ser Ser Lys Val Thr Ala Leu Ile Arg Met     770 775 780 Val Ala Leu Lys Lys Val Glu Leu Ile Ala Phe Ile Val 785 790 795 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 7 ggctttttat tctggaagat ag 22 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 8 gtttctgtct ctcccatata cagtc 25 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 9 aggctcagta agcaatgcgc 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 10 aacaaaagtg atgggaaatg ac 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 11 aactgtttgc tttctcctct gg 22 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 12 gcaagcctct cattttaccg 20 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 13 ttctttttct gttttcactt ggggca 26 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 14 acatctatta atctccttaa atgtccattc 30 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 15 tgaagtgaga agctaagaga actg 24 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 16 cgtgagaaat aaaaccaagt agagg 25 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 17 gacagaaggc tgtccttata a 21 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 18 catctgctta cgtgtatctt ca 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 19 tcttgaataa actgtgtagc gc 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 20 tccctatcac ctgtattcac c 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 21 tttccatttt ctgttagggg tg 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 22 agtcaatcag gaaaacatca gc 22 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 23 tttttggggg gaagtagcag 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 24 aacagagatc cctatgcagc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 25 gacacagtga gaccctatct 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 26 caccaacatc cacaaactgg 20 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 27 tgagatgttc ccactgacca at 22 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 28 caccatctac tctcccatca ctg 23 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 29 cagaatctca taggttgcca ataat 25 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 30 aacagcacca ccatatagca c 21 <210> 31 <211> 20 <212> DNA <213> Homo sapiens <400> 31 tacgaccaat gtaaacacat 20

[Brief description of drawings]

FIG. 1 is a photograph showing the results of Western blot analysis of wild-type GR and C643R mutant GR. Cytosolic fractions were prepared from COS-7 cells transfected with either vector, wild type or C643R mutant GR expression plasmid. This cytoplasmic fraction (50 μg) was electrophoresed and blotted.

FIG. 2: Wild-type GR and 2314 ins using cytoplasmic fraction
3 is a photograph showing the results of Western blot analysis of -a mutant GR. The cytoplasmic fraction can be vector, wild type or 2314 ins-a
Prepared from COS-7 cells transfected with either of the mutant GR expression plasmids. This cytoplasmic fraction (50 μ
g) was electrophoresed and blotted.

FIG. 3 is a photograph showing the results of Western blot analysis of wild-type GR and 2314 ins-a mutant GR using a lysate of whole cells. The whole cell lysate is a vector,
Prepared from COS-7 cells transfected with either wild type or 2314 ins-a mutant GR expression plasmid. This whole cell lysate (80 μg) was electrophoresed and blotted.

FIG. 4 is a diagram showing the results of measuring the transcriptional activity of wild type, C643R mutant GR and 2314 ins-a mutant GR. COS-
7 cells are vector, wild type GR, C643R mutant GR, or
Any of the 2314 ins-a mutant GRs were co-transfected with the MMTV promoter-luciferase reporter construct (pHH-Luc). Luciferase activity was measured after 18 hours incubation with dexamethasone.

FIG. 5 is a diagram showing analysis results of binding affinities of wild type and C643R mutant GR to dexamethasone. It represents one of the representative experiments. Wild type and C643R mutant GR
Was transiently expressed in COS-7 cells. Cells were harvested 48 hours post transfection and specific binding to [ ³ H] dexamethasone was analyzed by incubation of cytoplasmic fractions. This binding data was analyzed according to Scatchard plot analysis. Three separate binding assays were performed. The affinity of mutant GR was 1/6 that of wild type.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Junichi Sawada             1-18-1 Kamigaga, Setagaya-ku, Tokyo National doctor             Inside the Institute for Pharmaceutical and Food Hygiene (72) Inventor Shogo Ozawa             1-18-1 Kamigaga, Setagaya-ku, Tokyo National doctor             Inside the Institute for Pharmaceutical and Food Hygiene (72) Inventor Yoshiro Saito             1-18-1 Kamigaga, Setagaya-ku, Tokyo National doctor             Inside the Institute for Pharmaceutical and Food Hygiene F-term (reference) 4B024 AA11 BA63 CA01 CA04 CA06                       CA11 DA02 EA02 EA04 FA02                       FA10 GA11 GA18 HA01 HA12                       HA19                 4B063 QA05 QA13 QA17 QQ12 QQ42                       QR08 QR55 QR62 QS16 QS25                       QS39

Claims (22)

[Claims] 1. A method for testing the efficacy of a glucocorticoid preparation in a subject, which comprises decreasing the glucocorticoid-binding ability of a glucocorticoid receptor or reducing the transcription activation ability of the receptor. A test method comprising a step of detecting a polymorphism of a receptor gene, wherein the subject is determined to be resistant to the glucocorticoid preparation when the polymorphism is detected. 2. The test method according to claim 1, wherein the polymorphism is a single nucleotide polymorphism. 3. The test method according to claim 1, wherein the polymorphism in the glucocorticoid receptor gene causes a mutation at the 643th amino acid in the glucocorticoid receptor. 4. A polymorphism in the glucocorticoid receptor gene, which results in an amino acid mutation at the 643th amino acid in the glucocorticoid receptor, and 772 to 777 of the receptor.
The test method according to claim 1, which is a polymorphism that causes the th amino acid mutation. 5. The test method according to claim 3, wherein the 643rd amino acid mutation of the glucocorticoid receptor is a mutation of cysteine to arginine. 6. The polymorphism of the receptor gene that causes the amino acid mutation at the 643th amino acid in the glucocorticoid receptor is the polymorphism at the 1927th base site in the cDNA sequence of the gene. Inspection method. 7. The glucocorticoid receptor gene cd
The test method according to claim 6, wherein the polymorphism at the 1927th base site in the NA sequence is a mutation from thymine to cytosine. 8. A glucocorticoid receptor 772-7.
The test method according to claim 4, wherein the polymorphism of the receptor gene that causes the 77th amino acid mutation is a polymorphism at the 2314th base site in the cDNA sequence of the gene. 9. The glucocorticoid receptor gene cd
The test method according to claim 8, wherein the polymorphism at the 2314th base site in the NA sequence is a single-base insertion mutation in the site. 10. The test method according to claim 1, wherein the glucocorticoid-binding ability is a dexamezone-binding ability. 11. The method includes the following steps (a) to (d):
The inspection method according to claim 1. (A) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
Step (c) of isolating DNA containing the 927th base site or containing 1927th and 2314th base sites (c) Step of determining the base sequence of the isolated DNA (d) DNA determined by step (c) The base sequence of
Process to compare with control 12. The method includes the following steps (a) to (d):
The inspection method according to claim 1. (A) a step of preparing a DNA sample from a subject (b) a step of cleaving the prepared DNA sample with a restriction enzyme (c) a step of separating a DNA fragment according to its size (d) a detected DNA fragment Of comparing the size of 13. The method includes the following steps (a) to (e):
The inspection method according to claim 1. (A) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
Amplifying DNA containing the 927th base site, or containing the 1927th and 2314th base sites (c) Cleaving the amplified DNA with a restriction enzyme (d) Separation of DNA fragments according to their size Step (e) Comparing the size of the detected DNA fragment with a control 14. The method includes the following steps (a) to (e):
The inspection method according to claim 1. (A) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
A step of amplifying DNA containing the 927th base site, or containing the 1927th and 2314th base sites (c) Dissociating the amplified DNA into single-stranded DNAs (d) Dissociating the single-stranded DNAs Separation on non-denaturing gel (e) Comparison of mobility of separated single-stranded DNA on gel with control 15. The method includes the following steps (a) to (d):
The inspection method according to claim 1. (A) a step of preparing a DNA sample from a subject (b) a base site in the glucocorticoid receptor gene, which is 1 in the cDNA sequence of the receptor gene
Amplifying DNA containing the 927th base site or containing the 1927th and 2314th base sites (c) Separation of the amplified DNA on a gel in which the concentration of the DNA denaturant is gradually increased (d) Separation Comparing the mobility of immobilized DNA on a gel with a control 16. The method includes the following steps (a) to (c):
The inspection method according to claim 1. (A) (i) a base site in a glucocorticoid receptor gene prepared from a subject, including the 1927th base site in the cDNA sequence of the receptor gene, or the 1927th and 2314th base sites; And (ii) the DNA of step (a) (i) and step (a) (ii) of providing a substrate on which (ii) a nucleotide probe is immobilized.
Contacting the substrate of (c) by detecting the intensity of hybridization between the DNA and the nucleotide probe immobilized on the substrate,
Step of detecting polymorphism in gene encoding glucocorticoid receptor 17. A polymorphic site in the glucocorticoid receptor gene, which comprises at least 1927 polymorphic site in the cDNA sequence of the receptor gene.
A polynucleotide having a chain length of 5 nucleotides. 18. The polynucleotide according to claim 17, wherein the base at the polymorphic site is cytosine (C). 19. A polynucleotide which hybridizes to a DNA having a base site in the glucocorticoid receptor gene and containing the 1927th base site in the cDNA sequence of the receptor gene and having a chain length of at least 15 nucleotides. , A reagent for testing the effectiveness of glucocorticoid preparations. 20. Further, it is a base site in the glucocorticoid receptor gene, which is the cD of the receptor gene.
DNA containing the 2314th base site in the NA sequence
20. The reagent of claim 19, which comprises a polynucleotide that hybridizes to and has a chain length of at least 15 nucleotides. 21. A forward primer and a reverse primer designed to amplify a DNA containing a base site in the glucocorticoid receptor gene, the base site being the 1927th base position in the cDNA sequence of the receptor gene, A reagent for testing the effectiveness of glucocorticoid preparations. 22. Furthermore, it is a base site in the glucocorticoid receptor gene, which is the cD of the receptor gene.
DNA containing the 2314th base site in the NA sequence
22. The reagent according to claim 21, comprising a forward primer and a reverse primer designed to amplify A.