U.S. patent application number 15/112067 was filed with the patent office on 2017-01-05 for molecule associated with onset of gout, and method and kit for evaluating diathesis of uric acid-related diseases and inflammation-related diseases, and inspection object and drug.
The applicant listed for this patent is THE UNIVERSITY OF TOKYO. Invention is credited to Hirotaka Matsuo, Nariyoshi Shinomiya, Tappei Takada.
Application Number | 20170002413 15/112067 |
Document ID | / |
Family ID | 53543062 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170002413 |
Kind Code |
A1 |
Matsuo; Hirotaka ; et
al. |
January 5, 2017 |
MOLECULE ASSOCIATED WITH ONSET OF GOUT, AND METHOD AND KIT FOR
EVALUATING DIATHESIS OF URIC ACID-RELATED DISEASES AND
INFLAMMATION-RELATED DISEASES, AND INSPECTION OBJECT AND DRUG
Abstract
To specify a molecule associated with the onset of gout so as to
provide a method for evaluating a diathesis of uric acid-related
diseases and a diathesis of inflammation-related diseases, an
evaluation kit for carrying out the method, an inspection object,
and a drug, on the basis of the molecule specified above, for
contributing to the early treatment and prevention of the uric
acid-related diseases and inflammation-related diseases. The
molecule includes any one protein and cDNA of CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D and FAM35A, or proteins of combination thereof
with GLUT9, NPT1, URAT1, or NXRN2, and is capable of selectively
inducing gout. A molecule includes protein and cDNA of an ABCG2
variant and is capable of selectively and ATP-dependently
decreasing urate excretion.
Inventors: |
Matsuo; Hirotaka; (Saitama,
JP) ; Shinomiya; Nariyoshi; (Saitama, JP) ;
Takada; Tappei; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF TOKYO |
Tokyo |
|
JP |
|
|
Family ID: |
53543062 |
Appl. No.: |
15/112067 |
Filed: |
January 19, 2015 |
PCT Filed: |
January 19, 2015 |
PCT NO: |
PCT/JP2015/051232 |
371 Date: |
September 16, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/53 20130101;
A61K 38/1709 20130101; A61P 9/10 20180101; A61K 38/44 20130101;
A61P 9/00 20180101; G01N 2800/107 20130101; A61K 48/00 20130101;
A61P 25/00 20180101; A61P 19/02 20180101; A61K 38/00 20130101; C12Q
2600/156 20130101; A61K 38/45 20130101; A61P 13/12 20180101; A61K
38/177 20130101; A61P 9/06 20180101; G01N 33/6893 20130101; C07K
14/47 20130101; A61P 19/06 20180101; A61P 29/00 20180101; C12Y
207/11025 20130101; A61P 1/04 20180101; A61P 31/12 20180101; C12Q
1/6883 20130101; C12Y 102/01003 20130101; C12Y 603/04 20130101;
A61P 15/04 20180101; C12N 9/00 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 38/53 20060101 A61K038/53; A61K 38/45 20060101
A61K038/45; A61K 38/17 20060101 A61K038/17; A61K 38/44 20060101
A61K038/44 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2014 |
JP |
2014-006806 |
Sep 5, 2014 |
JP |
2014-181642 |
Claims
1. A molecule associated with onset of gout, comprising any one
protein or cDNA of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, ABCG2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, or
a combination thereof with any one protein or cDNA of GLUT9, NPT1,
URAT1, and NXRN2, and being capable of relating to the onset of
gout; or comprising protein or cDNA of an ABCG2 variant, and being
capable of selectively and ATP-dependently decreasing excretion of
urate.
2. A method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis, the method
comprising: evaluating whether or not a subject has a diathesis
capable of inducing urate regulation failure, or a state or a uric
acid-related disease attributable to the failure, and the
evaluating comprising: a step of detecting a gene polymorphism of a
gene encoding at least any one protein or cDNA of CNIH2-PACS1,
ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, or a combination thereof with gene
polymorphisms of GLUT9, NPT1, URAT1, and NXRN2, using a test sample
containing human genes of the subject.
3. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to claim 2,
wherein detection of a gene polymorphism of a gene encoding any one
protein or cDNA of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, ABCG2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, or
a combination thereof with GLUT9, NPT1, URAT1, and NXRN2 is
detection of a SNP or a polymorphism having a relationship of
linkage disequilibrium with the SNP or a polymorphism with a
frequency of 1% or less.
4. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to claim 2
or 3, wherein the method uses a combination with detection of SNPs
of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, in which the
SNPs are rs4073582, rs671, rs2188380, rs1260326, rs10791821,
rs56027330, rs11758351, rs4496782, and rs7903456, respectively, or
a gene polymorphism having a relationship of linkage disequilibrium
with the SNPs, or other gene polymorphisms.
5. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to any one
of claims 2 to 4, wherein the method uses detection of gene
polymorphisms of G279R of NPT4, and V178I, N299S, E311K, G462R,
V508I, V516M, A634V, F489L and D620G of ABCG2, or a gene
polymorphism having a relationship of linkage disequilibrium with
the polymorphisms, and a combination thereof.
6. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to any one
of claims 2 to 5, wherein the method uses a combination of
detection of a SNP of GLUT9 (rs3775948), a gene polymorphism (SNP)
of NPT1 (rs1165196, I269T), a SNP of URAT1 (rs505802), a SNP of
NXRN2 (rs2285340 or rs506338), or polymorphisms having a
relationship of linkage disequilibrium therewith, or W258X and R90H
of URAT1, and SNPs (Q126X, Q141K, and V12M) and polymorphisms
(R113X, F208S, G268R, P269S, E334X, S441N, L447V, S486N, F506SfsX4,
R575X, and C608X) of ABCG2.
7. A method for evaluating whether or not a subject has a diathesis
capable of inducing urate regulation failure, or a state or a
disease attributable to the failure, wherein the method is carried
out evaluation using a test sample containing human genes of the
subject, based on Q126X and Q141K of two SNPs of genes encoding
ABCG2 protein, and wherein when a gene encoding Q of Q126X is C/C
and a gene encoding Q of Q141K is C/C, the function of ABCG2 is
evaluated to be normal; when a gene encoding Q of Q126X is C/C, and
a gene encoding Q of Q141K is A/C, the function of ABCG2 is
evaluated to be 3/4; when a gene encoding Q of Q126X is T/C and a
gene encoding Q of Q141K is C/C, the function of ABCG2 is evaluated
to be 1/2; when a gene encoding Q of Q126X is C/C and a gene
encoding Q of Q141K is A/A, the function of ABCG2 is evaluated to
be 1/2; when a gene encoding Q of Q126X is T/C and a gene encoding
Q of Q141K is A/C, the function of ABCG2 is evaluated to be 1/4;
and when a gene encoding Q of Q126X is T/T and a gene encoding Q of
Q141K is C/C, ABCG2 is evaluated to have no function, and wherein
the method evaluates that a diathesis capable of inducing urate
regulation failure, or a state or a disease attributable to the
failure is evaluated to be high depending on a degree of loss of
the function of ABCG2.
8. A method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis, wherein when one
polymorphism which produces an amino acid variation of any one of
V178I, N299S, E311K, G462R, V508I, V516M, A634V, R113X, F208S,
G268R, E334X, S441N, S486N, and F506SfsX4 of ABCG2 is present, it
is evaluated that a subject has a diathesis capable of inducing
urate regulation failure or a state or a disease attributable to
the failure, substantially similar to the case in which one Q126X
is present.
9. A method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis, wherein when one
polymorphism which produces an amino acid variation of any one of
L447V, R575X, and C608X of ABCG2 is present, it is evaluated that a
subject has a diathesis capable of inducing urate regulation
failure or a state or a disease attributable to the failure,
substantially similar to the case in which one Q126X is
present.
10. A method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis, wherein presence of
one polymorphism which produces an amino acid variation of any one
of V12M, P269S, F489L, and D620G of ABCG2 is evaluated to be
associated with a diathesis capable of inducing urate regulation
failure or a state or a disease attributable to the failure.
11. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to any one
of claims 2 to 10, wherein the method evaluates estimation of
clinical disease types or suitable drugs based on the result
obtained by the method for evaluating a uric acid-related disease
diathesis and an inflammation-related disease diathesis as defined
in any one of claims 2 to 10.
12. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to any one
of claims 2 to 11, wherein when a serum uric acid level is a
predetermined value or more, it is evaluated that a subject has a
high diathesis capable of inducing urate regulation failure or a
state or a disease attributable to the failure.
13. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to claim
12, wherein a threshold of the serum uric acid level is in a range
from 6.0 to 9.0 mg/dl, and more preferably in a range from 7.0 to
8.0 mg/dl.
14. The method for evaluating a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to any one
of claims 2 to 13, wherein the uric acid-related disease and the
inflammation-related disease include hyperuricemia, gout,
rheumatoid arthritis, osteoarthritis, infertility, cerebral stroke,
neurodegenerative disease, ischemic heart disease, chronic kidney
disease, renal dysfunction, urolithiasis, kidney stone, aneurysm,
arrhythmia including atrial fibrillation, inflammatory bowel
disease, enteritis, functional dyspepsia, viral intestinal disease,
and photosensitivity.
15. An evaluation kit for a uric acid-related disease diathesis and
an inflammation-related disease diathesis, the kit evaluating
whether or not a subject has a diathesis capable of inducing urate
regulation failure, or a state or a disease attributable to the
failure, wherein the kit includes means for detecting a SNP of at
least any one gene selected from CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D,
and FAM35A genes, or a gene polymorphism having a relationship of
linkage disequilibrium with the SNP, or a polymorphism having a
frequency of 1% or less, or a combination thereof with a gene
polymorphism of GLUT9, NPT1, URAT1 and NXRN2, using a test sample
containing human genes of the subject.
16. The evaluation kit for a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to claim
15, wherein each SNP of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR,
MAP3K11, NPT4, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D and FAM35A is each of rs4073582, rs671,
rs2188380, rs1260326, rs10791821, rs56027330, rs3775948, rs1165196,
rs505802, rs2285340 or rs506338, rs11758351, rs4496782, and
rs7903456.
17. The evaluation kit for a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to claim 15
or 16, wherein detection in an ABCG2 gene is detection in at least
any one of V178I, N299S, E311K, G462R, V508I, V516M, A634V, Q126X,
Q141K, V12M, R113X, F208S, G268R, P269S, E334X, S441N, L447V,
S486N, F506SfsX4, R575X, C608X, F489L, and D620G, or combination
thereof.
18. An inspection object, which is a living body in which urate
transport kinetics is to be examined, the inspection object
comprising: a nonhuman animal having a deficiency of at least any
one gene selected from CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR,
MAP3K11, NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A; or a nonhuman
animal overexpressing or decreased-expressing at least any one gene
selected from human CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, or at least any one gene selected
from nonhuman CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
ABCG2, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A; a nonhuman animal overexpressing or
decreased-expressing a human ABCG2 gene or nonhuman human ABCG2
gene including at least any one polymorphism selected from V178I,
N299S, E311K, G462R, V508I, V516M, A634V, Q126X, Q141K, V12M,
R113X, F208S, G268R, P269S, E334X, S441 N, L447V, S486N, F506SfsX4,
R575X, C608X, F489L, and D620G of ABCG2, or a combination thereof;
a nonhuman cell line or a human cell line having a deficiency of at
least any one gene of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35, a nonhuman cell line or a human cell
line overexpressing or decreased-expressing at least any one gene
selected from human CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, or nonhuman CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, a nonhuman cell
line or a human cell line overexpressing or decreased-expressing
human ABCG2 gene or a nonhuman ABCG2 gene including at least any
one polymorphism selected from V178I, N299S, E311K, G462R, V508I,
V516M, A634V, Q126X, Q141K, V12M, P269S, R113X, F208S, G268R,
E334X, S441N, L447V, S486N, F506SfsX4, R575X, C608X, F489L, and
D620G of ABCG2, or combination thereof; or a cell membrane vesicle
prepared from the cell lines.
19. A drug for uric acid-related diseases and inflammation-related
diseases, for reducing a diathesis capable of inducing urate
regulation failure, or a state or a disease attributable to the
failure, the drug including polynucleotide or polypeptide encoding
at least any one protein selected from CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, or combination thereof with GLUT9,
NPT1, URAT1, and NXRN2 in a form capable of being introduced into
cells.
Description
TECHNICAL FIELD
[0001] The present invention relates to a molecule associated with
the onset of gout, as well as a method for evaluating a uric
acid-related disease diathesis and an inflammation-related disease
diathesis, and an evaluation kit for carrying out the method, and
also an inspection object and a drug relating to the method and
kit.
BACKGROUND ART
[0002] Recently, gout patients have increased and the onset age has
become younger. Gout is a disease caused by deposition of
monosodium urate crystals in tissue, and often has the onset as a
result of inflammation of the joint. Furthermore, gout is
frequently found in hyperuricemia patients, and has long been known
to have heritable components.
[0003] Gout is often complicated with hypertension, obesity,
diabetes, coronary artery diseases, cerebrovascular diseases,
kidney diseases, and the like. Furthermore, inflammation-related
diseases include rheumatoid arthritis, infertility, and the like.
Thus, early treatment and prevention of these diseases are
needed.
[0004] The present inventors have demonstrated, using
function-based genetic analysis, that loss-of-function type
mutations in two types of urate transporter genes, i.e., urate
transporter gene 1 (URAT1/SLC22A12) and glucose transporter 9
(GLUT9/SLC2A9), cause renal hypouricemia (MIM220150 and MIM612076,
respectively) (Non-Patent Literatures 1 and 2). These findings,
together with their renal expression patterns, also show that URAT1
and GLUT9 mediate renal urate reabsorption in human.
[0005] However, other urate transporters have not been identified
by such analyses, and urate transporters including pathogenenic
variants that increase the serum uric acid (SUA) level remain
unidentified.
[0006] The prior art relating to a urate transporter includes
Patent Literature 1, and the prior arts relating to ABCG2 as a
transporter include Patent Literatures 2 to 4. However, the prior
arts disclose the ABCG2 as a transporter of a drug, but not
disclose its involvement in urate transport.
[0007] Furthermore, it is generally known that the hyperuricemia
may be a risk of gout but does not necessarily cause gout, for
example, it is reported that the onset rate of gout in 5 years is
just about 20% even in patients having severe hyperuricemia having
a serum uric acid level of 9 mg/dL or more. However, it has not
been clarified what types of hyperuricemia patients develop gout,
and what types of hyperuricemia patients do not develop gout.
[0008] Therefore, in conventional medical treatment, patients
having not less than the predetermined level of hyperuricemia have
been prescribed with a urate lowering drug, although most of them
have low gout risk. On the other hand, some cases develop gout
although the serum uric acid level is not so high, and physical and
economic burden of the gout patients have been large.
[0009] In Patent Literature 5, the present inventors have disclosed
that ABCG2 (ATP-binding cassette G2) responsible for exporting
various drugs and endogenous compounds has had transported uric
acid at high affinity. Furthermore, the present inventors have
demonstrated for the first time that Q141K variation in the ABCG2
gene, which is frequently found in hyperuricemia or gout case,
decrease the transport activity to about half level; the transport
activity is lost in some variations including Q126X; in inspection
of an influence on uric acid levels of a Q141K polymorphism in
healthy subjects, the serum uric acid level is increased according
to the holding number of Q141K variations; and ABCG2 controls urate
excretion in the kidney, liver, and small intestine in human. That
is to say, the present inventors have found that the ABCG2 gene is
a major causative gene of gout.
[0010] This finding is a result supporting an established theory
that gout is a disease including unknown familial and genetic
factors, and is the first discovery in the world as an example
showing that common variants are pathogenic variants and cause a
common disease (gout).
[0011] Thus, providing medical care such as preventive medicine
with respect to cases of hyperuricemia and gout according to
individual differences is becoming possible. However, since about
20% of cases does not have variations in Q126X and Q141K of ABCG2,
development of more detailed genetic analysis technology has been
required. Furthermore, when variations are found, it is desirable
that specific measures, for example, a target values for diet be
set, but they have not been able to be made by conventional
technology.
CITATION LIST
Patent Literature
[0012] Patent Literature 1: JP-A-2003-93067, "Renal and placental
urate transporters and their genes".
[0013] Patent Literature 2: JP-A-2007-60967, "Detection method of
gene polymorphisms and screening method of drugs".
[0014] Patent Literature 3: JP-A-2004-16042, "Mutated
polynucleotides and nucleic acid molecules which can be used for
genetic diagnosis of abnormality in drug absorption involving ABCG2
protein".
[0015] Patent Literature 4: JP-A-2005-529618, "Prediction method of
drug transport capability by ABCG2 polymorphism". Non-Patent
Literatures
[0016] Patent Literature 5: Japanese Patent Application No.
2009-148106 "Urate Transporter, as well as Method and Kit for
Evaluating Urate Transport-Related Disease Factor and
Inflammation-Related Disease Factor, and Sample and Drug"
Non-Patent Literature
[0017] Non-Patent Literature 1: Enomoto A, Kimura H, Chairoungdua
A, et al. Molecular identification of a renal urate anion exchanger
that regulates blood urate levels. Nature 2002; 417: 447-52.
[0018] Non-Patent Literature 2: Matsuo H, Chiba T, Nagamori S, et
al. Mutations in glucose transporter 9 gene SLC2A9 cause renal
hypouricemia. Am J Hum Genet 2008; 83: 744-51.
[0019] Non-Patent Literature 3: Kondo C, Suzuki H, Itoda M, et al.
Functional analysis of SNPs variants of BCRP/ABCG2. Pharm Res 2004;
21: 1895-903.
[0020] Non-Patent Literature 4: Tin, A. et al. Genome-wide
association study for serum urate concentrations and gout among
African Americans identifies genomic risk loci and a novel URAT1
loss-of-function allele. Human Molecular Genetics 20, 4056-68
(2011).
[0021] Non-Patent Literature 5: Sulem, P. et al. Identification of
low-frequency variants associated with gout and serum uric acid
levels. Nature Genetics 43, 1127-30 (2011).
[0022] Non-Patent Literature 6: Kottgen, A. et al. Genome-wide
association analyses identify 18 new loci associated with serum
urate concentrations. Nature Genetics 45, 145-54 (2013).
[0023] Non-Patent Literature 7: Ichida, K. et al. Decreased
extra-renal urate excretion is a common cause of hyperuricemia.
Nature communications 3, 764 (2012).
[0024] Non-Patent Literature 8: Matsuo, H. et al. Common defects of
ABCG2, a high-capacity urate exporter, cause gout: a function-based
genetic analysis in a Japanese population. Sci Transl Med 1, 5ra11
(2009).
[0025] Non-Patent Literature 9: Matsuo, H. et al. Common
dysfunctional variants in ABCG2 are a major cause of early-onset
gout. Scientific Reports 3, 2014 (2013).
[0026] Non-Patent Literature 10: Hirotaka Matsuo, Kimiyoshi Ichida,
Tappei Takada, Akiyoshi Nakayama, Nariyoshi Shinomiya, Urate
transporter as predominant factor of uric acid regulation. Saibou
Kougaku, 31 (5), 553-557, 2012.
[0027] Non-Patent Literature 11: K. Maedaand Y. Sugiyama. Impact of
genetic polymorphisms of transporters on the pharmacokinetic,
pharmacodynamic and toxicological properties of anionic drugs. Drug
Metab Pharmacokinet. 23: 223-235 (2008).
[0028] Non-Patent Literature 12: C. Kondo, H. Suzuki, M. Itoda, S.
Ozawa, J. Sawada, D. Kobayashi, I. Ieiri, K. Mine, K. Ohtsubo, and
Y. Sugiyama. Functional analysis of SNPs variants of BCRP/ABCG2.
Pharm Res. 21: 1895-1903 (2004).
[0029] Non-Patent Literature 13: S. Koshiba, R. An, H. Saito, K.
Wakabayashi, A. Tamura, and T. Ishikawa. Human ABC transporters
ABCG2 (BCRP) and ABCG4. Xenobiotica. 38: 863-888 (2008).
[0030] Non-Patent Literature 14: A. Tamura, K. Wakabayashi, Y.
Onishi, M. Takeda, Y. Ikegami, S. Sawada, M. Tsuji, Y. Matsuda, and
T. Ishikawa. Re-evaluation and functional classification of
non-synonymous single nucleotide polymorphisms of the human
ATP-binding cassette transporter ABCG2. Cancer Sci. 98: 231-239
(2007).
[0031] Non-Patent Literature 15: Rohan S. Wijesurendra, Barbara
Casadei. Atrial Fibrillation: Effects Beyond the Atrium?
Cardiovascular Research Advance Access published Jan. 12, 2015.
[0032] Non-Patent Literature 16: Muhammad A. Balouch, Matthew J.
Kolek, Dawood Darbar. Improved understanding of the pathophysiology
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2014: 5.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0033] Accordingly, the object of the present invention is to
provide a method for evaluating a uric acid-related disease
diathesis and an inflammation-related disease diathesis and to
provide an evaluation kit for carrying out the method, and an
inspection object and a drug relating to the method and the kit so
that a high-capacity urate transporter is identified so as to
contribute to early treatment and prevention of uric acid-related
diseases and inflammation-related diseases on the basis of the
identified transporter.
Solution to Problem
[0034] A molecule associated with the onset of gout of the present
invention is a molecule which is associated with the onset of gout,
and is characterized by including any one protein or cDNA of
CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, or a
combination thereof with any one protein or cDNA of GLUT9, NPT1,
URAT1, and NXRN2, and being capable of relating to the onset of
gout; or including protein or cDNA of an ABCG2 variant, and being
capable of selectively and ATP-dependently decreasing excretion of
urate.
[0035] A method for evaluating a uric acid-related disease
diathesis and an inflammation-related disease diathesis of the
present invention includes evaluating whether or not a subject has
a diathesis capable of inducing urate regulation failure, or a
state or a uric acid-related disease attributable to the failure.
The evaluating includes a step of detecting a gene polymorphism of
a gene encoding at least any one protein or cDNA of CNIH2-PACS1,
ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, or a combination thereof with gene
polymorphisms of GLUT9, NPT1, URAT1, and NXRN2, using a test sample
containing human genes of the subject.
[0036] Furthermore, the detection of a gene polymorphism of a gene
encoding any one protein or cDNA of CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D,
and FAM35A, or a combination thereof with GLUT9, NPT1, URAT1, and
NXRN2 may be the detection of a SNP or a polymorphism having a
relationship of linkage disequilibrium with the SNP or a
polymorphism with a frequency of 1% or less.
[0037] Herein, a combination with detection of SNPs of CNIH2-PACS1,
ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A (rs4073582, rs671, rs2188380,
rs1260326, rs10791821, rs56027330, rs11758351, rs4496782, and
rs7903456, respectively), or a gene polymorphism having a
relationship of linkage disequilibrium with the SNPs, or other gene
polymorphisms may be used.
[0038] Furthermore, detection of gene polymorphisms of G279R of
NPT4, and V178I, N299S, E311K, G462R, V508I, V516M, A634V, F489L,
and D620G of ABCG2, or a gene polymorphism having a relationship of
linkage disequilibrium with the polymorphisms, and a combination
thereof may be used.
[0039] A combination of detection of a SNP of GLUT9 (rs3775948),
gene polymorphisms (SNPs) of NPT1 (rs1165196 and I269T), a SNP of
URAT1 (rs505802), a SNP of NXRN2 (rs2285340 or rs506338), or
polymorphisms having a relationship of linkage disequilibrium
therewith, or W258X and R90H of URAT1, and SNPs (Q126X, Q141K, and
V12M) and polymorphisms (R113X, F208S, G268R, P269S, E334X, S441N,
L447V, S486N, F506SfsX4, R575X, and C608X) of ABCG2 may be
used.
[0040] Furthermore, in evaluation using a test sample containing
human genes of the subject, based on Q126X and Q141K of two SNPs of
genes encoding ABCG2 protein, when a gene encoding Q of Q126X is
C/C and a gene encoding Q of Q141K is C/C, the function of ABCG2 is
evaluated to be normal; when a gene encoding Q of Q126X is C/C, and
a gene encoding Q of Q141K is A/C, the function of ABCG2 is
evaluated to be 3/4; when a gene encoding Q of Q126X is T/C and a
gene encoding Q of Q141K is C/C, the function of ABCG2 is evaluated
to be 1/2; when a gene encoding Q of Q126X is C/C and a gene
encoding Q of Q141K is A/A, the function of ABCG2 is evaluated to
be 1/2; when a gene encoding Q of Q126X is T/C and a gene encoding
Q of Q141K is A/C, the function of ABCG2 is evaluated to be 1/4;
and when a gene encoding Q of Q126X is T/T and a gene encoding Q of
Q141K is C/C, ABCG2 is evaluated to have no function. The method
may evaluate that a diathesis capable of inducing urate regulation
failure, or a state or a disease attributable to the failure is
evaluated to be high depending on a degree of loss of the function
of ABCG2.
[0041] Furthermore, when one polymorphism which produces an amino
acid variation of any one of V178I, N299S, E311K, G462R, V508I,
V516M, A634V, R113X, F208S, G268R, E334X, S441N, S486N, and
F506SfsX4 of ABCG2 is present, it may be evaluated that a subject
has a diathesis capable of inducing urate regulation failure or a
state or a disease attributable to the failure, substantially
similar to the case in which one Q126X is present.
[0042] When one polymorphism which produces an amino acid variation
of any one of L447V, R575X, and C608X of ABCG2 is present, it may
be evaluated that a subject has a diathesis capable of inducing
urate regulation failure or a state or a disease attributable to
the failure, substantially similar to the case in which one Q126X
is present.
[0043] Presence of one polymorphism which produces an amino acid
variation of any one of V12M, P269S, F489L, and D620G of ABCG2 may
be evaluated to be associated with a diathesis capable of inducing
urate regulation failure or a state or a disease attributable to
the failure.
[0044] Furthermore, the method may evaluate estimation of clinical
disease types or suitable drugs based on the result obtained by the
method for evaluating a uric acid-related disease diathesis and an
inflammation-related disease diathesis mentioned above.
[0045] When a serum uric acid level is a predetermined value or
more, it may be evaluated that a subject has a high diathesis
capable of inducing urate regulation failure or a state or a
disease attributable to the failure.
[0046] A suitable threshold of the serum uric acid level is in a
range from 6.0 to 9.0 mg/dl, and more preferably in a range from
7.0 to 8.0 mg/dl.
[0047] Examples of the uric acid-related diseases and
inflammation-related diseases include hyperuricemia, gout,
rheumatoid arthritis, osteoarthritis, infertility, cerebral stroke,
neurodegenerative disease, ischemic heart disease, chronic kidney
disease, renal dysfunction, urolithiasis, kidney stone, aneurysm,
arrhythmia including atrial fibrillation, inflammatory bowel
disease, enteritis, functional dyspepsia, viral intestinal disease,
and photosensitivity.
[0048] The evaluation kit for uric acid-related disease diathesis
and inflammation-related disease diathesis according to the present
invention is a kit for evaluating whether or not a subject has a
diathesis capable of inducing urate regulation failure, or a state
or a disease attributable to the failure, wherein the kit includes
means for detecting a SNP of at least any one gene selected from
CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A genes, or a gene
polymorphism having a relationship of linkage disequilibrium with
the SNP, or a polymorphism having a frequency of 1% or less, or a
combination thereof with a gene polymorphism of GLUT9, NPT1, URAT1,
and NXRN2, using a test sample containing human genes of the
subject.
[0049] Herein, for the SNP of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR,
MAP3K11, NPT4, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, rs4073582, rs671, rs2188380,
rs1260326, rs10791821, rs56027330, rs3775948, rs1165196, rs505802,
rs2285340, or rs506338, rs11758351, rs4496782, and rs7903456 can be
used.
[0050] Furthermore, detection of ABCG2 genes may include detection
of at least any one of or combination of V178I, N299S, E311K,
G462R, V508I, V516M, A634V, F489L, D620G, Q126X, Q141K, V12M,
R113X, F208S, G268R, P269S, E334X, S441N, L447V, S486N, F506SfsX4,
R575X, and C608X.
[0051] The inspection object of the present invention is a living
body in which urate transport kinetics is to be examined. The
inspection object includes: a nonhuman animal having a deficiency
of at least any one gene selected from CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A; or a nonhuman
animal overexpressing or decreased-expressing at least any one gene
selected from human CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, or at least any one gene selected
from nonhuman CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
ABCG2, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A; a nonhuman animal overexpressing or
decreased-expressing a human ABCG2 gene or nonhuman human ABCG2
gene including at least any one polymorphism selected from V178I,
N299S, E311K, G462R, V508I, V516M, A634V, F489L, D620G, Q126X,
Q141K, V12M, R113X, F208S, G268R, P269S, E334X, S441N, L447V,
S486N, F506SfsX4, R575X, and C608X of ABCG2, or a combination
thereof; a nonhuman cell line or a human cell line having a
deficiency of at least any one gene of CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35; a nonhuman cell
line or a human cell line overexpressing or decreased-expressing at
least any one gene selected from human CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, GLUT9, NPT1, URAT1, NXRN2,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, or nonhuman
CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, ABCG2, GLUT9,
NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and
FAM35A; a nonhuman cell line or a human cell line overexpressing or
decreased-expressing human ABCG2 gene or a nonhuman ABCG2 gene
including at least any one polymorphism selected from V178I, N299S,
E311K, G462R, V508I, V516M, A634V, F489L, D620G, Q126X, Q141K,
V12M, P269S, R113X, F208S, G268R, E334X, S441N, L447V, S486N,
F506SfsX4, R575X, and C608X of ABCG2, or combination thereof; or a
cell membrane vesicle prepared from the cell lines.
[0052] A drug for uric acid-related diseases and
inflammation-related diseases of the present invention is a drug
for uric acid-related diseases and inflammation-related diseases,
for reducing a diathesis capable of inducing urate regulation
failure, or a state or a disease attributable to the failure. The
drug includes polynucleotide or polypeptide encoding at least any
one protein selected from CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR,
MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and
FAM35A, or combination thereof with GLUT9, NPT1, URAT1, and NXRN2
in a form capable of being introduced into cells.
Advantageous Effects of Inventions
[0053] The present invention contributes to early treatment and
prevention of diseases related to uric acid regulation.
[0054] With the improvement of a nutritional state, hyperuricemia
continues to increase. Although only a part of hyperuricemias that
advances to gout, many patients with hyperuricemias undergo
treatment with a urate lowering drug regardless of the onset of
gout. On the contrary, the present invention can previously obtain
information--such as loss or decrease in function of gout-related
genes, which lead to identification of patients who should
preferentially start treatment, reduction of medical care
expenditure of the urate lowering drug and the like, and reduction
of physical and economic burdens of persons who are likely to
develop gout. Furthermore, the present invention contributes to
measurement of effects of various types of medication because a
gout-related gene transports anti-cancer drugs, therapeutic agents
for various types of lifestyle-related diseases, and the like.
Furthermore, the present invention enables specific measures for
lifestyle, for example, setting of target values for diet to be
set, and therefore, contributes to early prevention and early
treatment of individual cases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1 is a Manhattan plot graph of the genome-wide
association study of all gouts.
[0056] FIG. 2 is a Manhattan plot graph of the genome-wide
association study of a ROL type grout.
[0057] FIG. 3 is a Manhattan plot graph of the genome-wide
association study of a RUE type grout.
[0058] FIG. 4 is a graph showing a genomic region of ABCG2,
including genome-wide significant association.
[0059] FIG. 5 is a graph showing a genomic region of MYL2-CUX2,
including genome-wide significant association.
[0060] FIG. 6 is a graph showing a genomic region of SLC2A9,
including genome-wide significant association.
[0061] FIG. 7 is a graph showing a genomic region of GCKR,
including genome-wide significant association.
[0062] FIG. 8 is a graph showing a genomic region of CNIH2-PACS1,
including genome-wide significant association.
[0063] FIG. 9 is a graph showing a genomic region of MAP3K11.
[0064] FIG. 10 is a table showing five SNPs associated with gout at
a genome-wide significant level and one suggestive SNP.
[0065] FIG. 11 is an explanatory diagram showing classification of
gout.
[0066] FIG. 12 is a table showing the relationship between seven
SNPs and subtypes of gout and urate-transport parameters which the
subtypes are based on.
[0067] FIG. 13 shows graphs each showing an influence of risk
alleles of the identified SNPs on urate transport-related clinical
parameters.
[0068] FIG. 14 is an explanatory diagram showing an influence of
difference in SNPs on the disease types of gout and
hyperuricemia.
[0069] FIG. 15 is a table showing analysis results of the
association between gout and tag SNPs of MYL2-CUX2 locus, as well
as effects of analysis results of tag SNPs with adjustment by
rs671.
[0070] FIG. 16 is a table showing analysis results of the
association between gout and ALDH2 gene rs671 or alcohol
drinking.
[0071] FIG. 17 is a graph showing additive effects of six
gout-related SNPs causing the onset of gout.
[0072] FIG. 18 is an explanatory diagram showing clinical
classification of hyperuricemia and gout.
[0073] FIG. 19 is an explanatory diagram showing haplotypes of
ABCG2.
[0074] FIG. 20 is an explanatory diagram showing an onset risk by
ABCG2 dysfunction in each type of gout.
[0075] FIG. 21 is a table showing frequency of ABCG2 function in
Japanese individuals.
[0076] FIG. 22 is an explanatory diagram showing PAR % (Population
attributable risk percent) of hyperuricemia by ABCG2
dysfunction.
[0077] FIG. 23 is an explanatory diagram showing a significant
increase of the serum uric acid level by ABCG2 dysfunction.
[0078] FIG. 24 is a table showing effects of ABCG2 dysfunction,
BMI, alcohol intake, and the like, on the serum uric acid
level.
[0079] FIG. 25 is an explanatory diagram showing a transport system
of urate.
[0080] FIG. 26 is an explanatory diagram showing the relationship
between ABCG2 dysfunction and the onset risk of gout.
[0081] FIG. 27 is an explanatory diagram showing a structure and
variations of ABCG2.
[0082] FIG. 28 is an explanatory diagram showing the relationship
between function of ABCG2 and the onset risk of gout for each
age.
[0083] FIG. 29 is an explanatory diagram showing disease type
classification of hyperuricemia.
[0084] FIG. 30 is an explanatory diagram showing positions of seven
types of amino acid variations of ABCG2.
[0085] FIG. 31 is a graph and western blotting photographs showing
quantitation of mRNA and protein of the wild type and variant ABCG2
in HEK293 cells.
[0086] FIG. 32 shows confocal microphotographs showing
intracellular localization in the wild type and variant ABCG2 in
LLC-PK1 cells.
[0087] FIG. 33 is a western blotting photograph of protein
quantitation using a cell membrane vesicle expressing the wild type
and variant ABCG2.
[0088] FIG. 34 is a graph showing urate transport by an ABCG2
variant.
[0089] FIG. 35 is a table showing polymorphisms and variations of
ABCG2.
[0090] FIG. 36 is a table showing the results of analysis of the
relationship between gout and a gene polymorphism rs1165196 of
NPT1/SLC17A1.
[0091] FIG. 37 is a photograph showing localization of NPT1 in
human kidney by immunohistochemical staining.
[0092] FIG. 38 is a diagram showing a physiological function of
NPT1.
[0093] FIG. 39 shows a graph and a western blotting photograph
showing the results of urate transport analysis of mutated
ABCG2.
[0094] FIG. 40 is a graph showing nonsynonymous variants of the
ABCG2 gene found in the sequence analysis of gout cases.
[0095] FIG. 41 is a table showing the results of analysis of the
relationship between hyperuricemia and URAT1 nonsynonymous
variants.
[0096] FIG. 42 is a table showing a result of genome-wide
association study of gout after replication analysis using a custom
chip.
[0097] FIG. 43 is a table showing the results of analysis of the
change of urinary coproporphyrin based on the function of
ABCG2.
[0098] FIG. 44 is a table showing the results of analysis of the
relationship between the function of ABCG2 and cerebral stroke.
[0099] FIG. 45 is a table showing the results of analysis of the
serum uric acid levels in ulcerative colitis case based on the
function of ABCG2.
[0100] FIG. 46 is a table showing the results of analysis of the
serum uric acid level before treatment of viral enteritis cases
based on the function of ABCG2.
[0101] FIG. 47 is a table showing the results of analysis of the
relationship between function of ABCG2 and ages at which dialysis
is introduced and the serum uric acid levels in hemodialysis
cases.
[0102] FIG. 48 is a table showing the results of analysis of the
relationship between the function of ABCG2 and the onset age of
gout and Parkinson's disease.
[0103] FIG. 49 is an explanatory diagram showing differential
effects of ABCG2 dysfunction on gout and Parkinson's disease.
DESCRIPTION OF EMBODIMENTS
[0104] The present inventors have found a high-capacity transporter
of urate as an extension of the findings disclosed in Non-Patent
Literatures 1 to 2 and 5, and the like, and thus reached the
present invention.
[0105] The present invention will be described below by showing
demonstration experiments constituting the basis of the present
invention. Embodiments of the present invention are not limited to
the below-mentioned Examples, and design can be changed by
appropriately using conventionally known techniques.
[0106] Although Japanese individuals are mainly described as the
subject herein, the present invention can be similarly applied to
other races. This is also based on the background that it is known
that the prevalence of gout is high in the Pacific Rim population
including Taiwanese aborigines, and the gene noted in the present
invention, ABCG2, is present in a gene region on the long arm of
the fourth chromosome found by a linkage study of 21 pedigrees in
Taiwan with the onset of gout.
[0107] The ATP-binding cassette, subfamily G, member 2 gene
ABCG2/BCRP locates in a gout-susceptibility locus (MIM138900) on
chromosome 4q, and it has a function of encoding a multispecific
transporter that is expressed on the apical membrane in several
tissues including intestine, liver, and kidney. Also, ABCG2 is a
transporter of nucleotide analogues that are structurally similar
to urate (Non-Patent Literature 3).
[0108] From GWAS at serum uric acid level, gene loci including
those of SLC2A9 and ABCG2 have been identified, and subsequent
genetic and functional studies have clarified biological and
pathological importance of ABCG2 encoding an urate exporter as a
major genetic risk of gout.
[0109] GWAS of gout was carried out three times. All the GWAS
includes self-stating gout patients (Non-Patent Literatures 4 to
6), and provided poor clinical information. In order to understand
genetic basis of gout more sufficiently, the present inventors
carried out GWAS of gout using only clinically defined cases for
the first time. In addition, the relationship between genetic
variation and gout subtype was evaluated based on the
urate-transport parameters, they have clarified genetic
heterogeneity of the gout subtype.
[0110] Participants of this time GWAS include 946 clinically
defined Japanese male patients and 1213 male controls. All samples
were genotyped by using Illumina, and then strict quality control
filtering was carried out. Based on the analysis of main
components, one gout patient estimated to be a hybrid of East Asian
race and Europe race was excluded.
[0111] FIGS. 1 to 3 are Manhattan plot graphs respectively showing
genome-wide association studies with respect to all gout, ROL type,
and RUE type. X-axis shows chromosome positions; Y-axis shows
-log.sub.10 P-values. The horizontal black dotted line indicates
the genome-wide significance threshold and a gray dotted line
indicates the cut-off level for selecting SNP for replication study
(P=5.0.times.10.sup.-8), respectively.
[0112] FIGS. 4 to 9 are graphs showing five genomic regions
including genome-wide significant association, respectively showing
plots of ABCG2, MYL2-CUX2, SLC2A9, GCKR, CNIH2-PACS1, and MAP3K11.
They show regions within 250 Kb from an SNP showing the minimum
P-value. The upper panel shows a plot of -log.sub.10 P-values
related to the test of the relationship between the SNP and gout,
and shows the SNP having the minimum P-value by pink-colored
diamonds. The other SNPs are classified by colors according to the
degree of the level linkage disequilibrium with the SNP showing the
minimum P-value (evaluated by r.sup.2). The center panel shows
plotting of recombinant frequency (centimorgan/Mb) estimated from
the phase II data of HapMap are plotted. The lower panel is a
RefSeq gene, and the genomic locus is based on the Genome Reference
Consortium GRCh37.
[0113] In the GWAS stage, SNPs were specified at three gene luci
showing evidence of genome-wide association at a significant level.
As shown in FIGS. 1 and 4 to 6, the SNPs were rs2728125 of ABCG2
(P=1.5.times.10.sup.-27; odds ratio [OR]=2.05), rs3775948 of SLC2A9
(P=6.7.times.10.sup.-15; odds ratio [OR]=1.64), and rs2188380 of
MYL2-CUX2 (P=5.7.times.10.sup.-13; odds ratio [OR]=1.78). In order
to identify risk gene loci by validating these gene loci,
independent samples of 1048 cases and 1334 controls were employed.
In the GWAS stage, among 124 SNPs showing the association in the
range of P<5.0.times.10.sup.-9, 17 SNPs were selected for a
replication analysis with the linkage disequilibrium considered.
The genotypes of these 17 SNPs were determined by TaqMan assay.
[0114] FIG. 10 is a table showing five SNPs associated with gout at
a genome-wide significant level and one suggestive SNP. "Chr."
represents chromosome, "Freq." represents frequency of A1, "OR"
represents an odds ratio, and "CI" represents confidence interval.
In ".sup.adbSNP rs number", a suggestive SNP is marked with
".dagger.". ".sup.b": Positions of SNPs are based on NCBI human
genome reference sequence Buid 37.4; ".sup.c": A1 represents
risk-associated allele and A2 represents non-risk-associated
allele. ".sup.d": 945 gout cases and 1,213 controls. ".sup.e": 1,
048 gout cases and 1,334 controls. ".sup.f": Meta-analysis
combining GWAS and replication samples (1,993 gout cases and 2,547
controls).
[0115] In the GWAS stage, in three SNPs (rs2728125 of ABCG2,
rs3775948 of SLC2A9, and rs2188380 of MYL2-CUX2) that are beyond
genome-wide significant threshold, appropriate reproducibility was
obtained.
[0116] Furthermore, two SNPs (rs1260326 of GCKR and rs4073582 of
CNIH2-PACS1) showed significant association with gout at
P<2.9.times.10.sup.-3 (17 tests were adjusted by the Bonferroni
correction).
[0117] As shown in FIGS. 5 to 7, and 10, all of these five SNPs
reached genome-wide significance in the meta-analysis composed of
GWAS and a replication experiment (rs1260326 of GCKR
(P.sub.meta=1.9.times.10.sup.-12; OR=1.36), rs4073582 of
CNIH2-PACS1 (P.sub.meta=6.4.times.10.sup.-9; OR=1.66), and intron
SNP (rs10791821) of MAP3K11 showed association at a suggestive
level (P.sub.meta=1.0.times.10.sup.-7; OR=1.57).
[0118] The discovered five risk gene loci include a plurality of
genes responsible for-metabolic pathways. ABCG2 and SLC2A9 are
well-known urate transporter genes associated with the serum uric
acid level and gout. The present inventors formerly showed that two
nonsynonymous SNPs of ABCG2, that is, rs72552713 (Gln126Ter) and
rs2231142 (Gln141Lys), are strongly associated with hyperuricemia
and gout (Non-Patent Literatures 7 to 9). The risk alleles of these
two SNPs are present on different haplotypes. Also in GWAS, SNP
(rs2728125) having the highest significant association was in a
strong LD state with rs2231142 (r.sup.2=0.755). Multivariate
logistic regression analysis including these three SNPs of ABCG2
shows that rs2728125 no longer shows significant association
(P=0.19), but two nonsynonymous SNPs, that is, rs72552713 and
rs2231142, remain highly significant. This suggests that rs2728125
is just a sarrogate marker for true causal nonsynonymous
variant.
[0119] The frequency of allele (Glu504Lys) of rs671 of ALDH2 is
different among populations, this Glu 504Lys allele is general in
East Asian populations including Japanese population, but extremely
rare in the other populations such as European or African descent.
Therefore, in the GWAS of gout in European and African American,
SNP is not likely to be detected because of low frequency. ALDH2 is
an important gene in alcohol metabolism, and alcohol metabolism
covert acetaldehyde into acetic acid by oxidization in the
degradation process of alcohol. The Glu 504Lys allele decreases the
enzymatic activity of ALDH2. Recent study shows that rs671 is
associated with alcohol drinking behavior, and the alcohol drinking
is well-known to be a risk factor of gout.
[0120] GCKR suppresses glucokinase (GCK) which is an important
enzyme for glucose metabolism during fasting.
[0121] The gout risk allele of rs1260326 is associated with the
increase in the level of triglyceride and serum uric acid. Further,
its association with dyslipidemia is also reported. The present
GWAS is the first report to show that a common missense variant of
GCKR is associated with gout at a genome-wide significant
level.
[0122] It is known that CNIH2-PACS1 modulates the function of a
glutamate receptor of AMPA-subtype (AMPAR). CNIH2-PACS1 amplifies
the surface expression of AMPAR, and modulates gating thereof.
Furthermore, CNIH2-PACS1 mediates synaptic transmission of AMPAR in
hippocampus. At the same time, rs4073582 of CNIH2-PACS1 was in a
strong LD state with rs801733 of phosphofurin acidic cluster
sorting protein 1 which was associated with severe obesity
(r.sup.2=0.966) (FIG. 8). Therefore, also PACS1 can be a highly
susceptible gene as a good candidate gene.
[0123] A suggestive level association was detected between
rs10791821 of MAP3K11 and gout. This SNP was associated with the
expression level of MAP3K11 in the monocyte
(P=6.95.times.10.sup.-17). Further investigation is needed in order
to determine association with gout. This finding would be a clue to
a new molecule mechanism of gout. MAP3K11 is a member of MAP3K
super family, and is known to activate c-Jun N-terminal kinase
(JNK) that is a protein kinase activated by stress. Interestingly,
this JNK pathway is activated by phagocytosis of MSU crystal by
monocyte and macrophage, thus causing gouty arthritis.
[0124] FIG. 11 is an explanatory diagram showing classification of
gout. Genetic heterogeneity of gout subtypes was examined based on
ROL type gout and RUE type gout having different features based on
the amount of uric acid clearance (FE.sub.UA) and kidney urate
excretion (UUE) (Non-Patent Literature 7). Subtype-specific GWAS
showed apparent differences in association signals (FIGS. 2 to 3),
ROL and RUE type-specific GWAS showed peak signals on ABCG2 and
SLC2A9, respectively. On the other hand, an association signal in
MYL2-CUX2 did not show difference between these subtypes. Whether
or not the degree of association is different depending on subtypes
between the identified SNPs and gout was examined by an analysis of
subtype-specific OR and a case-subtype heterogeneity test.
According to this subgroup analysis, the association of two
nonsynonymous SNPs (rs72552713 and rs2231142) of ABCG2 is stronger
in the ROL type (OR=4.35 and 3.37) than in the RUE type (OR=1.28
and 1.88, respectively). The difference in OR between these
subtypes were extremely significant (P=2.4.times.10.sup.-5 and
1.0.times.10.sup.-7). On the other hand, association of rs3775948
of SLC2A9 was stronger in the RUE type (OR=1.94) than in the ROL
type (OR=1.38). The case-subtype heterogeneity test showed
significant difference in OR (P=2.7.times.10.sup.-4). The other
SNPs did not show significant differences in OR between
subtypes.
[0125] FIG. 12 is a table showing the relationship between seven
SNPs and subtypes of gout and urate-transport parameters which the
subtypes are based on, respectively. "FE.sub.UA" represents an
excretion rate of urate clearance (unit: %), "UUE" represents
urinary urate excretion (unit: mg/hour/1.73 m.sup.2), "ROL"
represents renal overload, "RUE" represents renal underexcretion,
"Coef." represents regression coefficient, "OR" represents an odds
ratio, and "CI" represents a confidence interval. ".sup.a": dbSNP
rs number. ".sup.b": the present inventors performed multivariate
logistic regression analyses, in which all the seven SNPs, alcohol
drinking and BMI were included in the model; 1,613 gout patients
and 1,334 controls with genotypes for rs72552713 and rs2231142 of
ABCG2, which were not on illumina OmniExpress platform, were used,
and 375 and 509 gout patients were grouped into sub-phenotypes,
that is, the ROL type and the RUE type, respectively. "c": P-values
of less than 0.05 were shown in bold letters.
[0126] FIG. 13 shows graphs showing a function of natural logarithm
of OR in a case-subtype heterogeneity test, showing the effects of
the risk allele of identified SNPs on the clinical parameters of
urate transport. (A) shows FE.sub.UA, and (B) shows UUE. OR in the
case-subtype heterogeneity test represents an estimated value of
the ratio of case-subtype OR in the ROL type and the RUE type. OR
is a value of more than 1 when SNP has a stronger effect in the ROL
type than in the RUE type. Diamonds and straight lines represent
point estimated values and 95% CI thereof. Pearson correlation
coefficient (r) and significant variance of the coefficient from
zero were examined.
[0127] Association between SNPs and the urate-transport parameter
(FE.sub.UA and UUE) was evaluated. Only SNP showing a significant
difference in OR between subtypes was significantly associated with
these two clinical parameters, risk alleles of the two SNPs of
ABCG2 and rs3775948 of SLC2A9 were associated with increase and
decrease in FE.sub.UA and UUE levels, respectively. When the effect
of the risk allele of each SNP with respect to urate-transport
parameter was plotted, OR in the case-subtype heterogeneity test
(estimation value of the case-control OR with respect to the
subtypes) as a function of the natural logarithm, clear straight
line relation was shown (r=0.96 [P=5.0.times.10.sup.-4] for
FE.sub.UA, and r=0.96 [P=4.8.times.10.sup.-4] for UUE).
[0128] This result shows that alleles strongly associated with the
risk of gout exhibits a differential effect on the urate-transport
parameters, and leads to the onset of particular subtype of gout.
Furthermore, this result takes extraordinary effects on the urate
excretion pathways into consideration. Decrease of FE.sub.UA and
UUE by the risk allele of SLC2A9 reflects dysfunction of the renal
urate excretion pathway. The increase of FE.sub.UA and UUE by the
risk allele of the two SNPs of ABCG2 observed herein can be
explained by the overload effect on renal excretion as compensation
of the dysfunction of intestinal excretion pathway due to failure
in ABCG2 function. The result is consistent with the findings
obtained from ABCG2 knockout mice (Non-Patent Literature 7).
Analysis of the gout subtype and the clinical parameters of the
urate transport showed the genetic heterogeneity of the gout
subtype and continuous distribution in the effective amount. The
significant heterogeneity in OR between the subtypes of SNPs of
ABCG2 and SLC2A9 having the strongest effect on the gout subtypes
and the clinical parameters was detected. Subtype-specific GWAS and
a subsequent replication analysis can be useful for identification
of genetic factors that are associated with only the particular
gout subtype.
[0129] When the association of rs671 with gout was analyzed in
which adjustment with alcohol drinking was carried out, it is shown
that the association of rs671 remains highly significant even after
adjustment with alcohol drinking (OR=1.62; P=6.5.times.10.sup.-9).
This shows the importance of rs671 in evaluation of the risk of the
gout.
[0130] FIG. 14 is an explanatory diagram showing effects of
differences in SNPs on the disease types of gout and hyperuricemia;
FIGS. 15 and 16 are tables showing the results of analysis of
association between gout and tag SNPs of MYL2-CUX2 locus, performed
with respect to 1048 male gout patients and 1334 controls, as well
as the results of analysis of the effects of tag SNPs by adjustment
with rs671; and a table showing analysis results of the association
between gout and ALDH2 gene rs671 or alcohol drinking, showing
results of analysis performed with respect to 1048 male gout
patients and 1323 controls (subjects having information of alcohol
drinking), respectively. FIG. 17 is a graph showing additive
effects of six gout-related SNPs causing the onset of gout, showing
results of analysis performed with respect to 1993 male gout
patients and 1334 controls (subjects having information of six
SNPs).
[0131] The analyses shown in FIGS. 15 and 16 showed significant
relationship between gene polymorphism rs671 of ALDH2 gene (alcohol
metabolism-related gene) and gout. In particular, FIG. 16 shows
that association between rs671 of ALDH2 gene and gout is
significant even after adjustment with alcohol drinking.
[0132] The analysis shown in FIG. 17 showed that, by counting the
number of risk alleles in each sample, and cumulative effects of
SNPs (rs72552713, rs2231142, rs671, rs3775948, rs1260326, and
rs4073582) at the identified gene loci were evaluated. When
reference category was set to 4 or less risk alleles, ORs for gout;
to achieve 5, 6, 7, 8, and 9 or more risk alleles were 2.18, 3.83,
6.51, 12.8, and 24.9, respectively.
[0133] FIG. 18 is an explanatory diagram showing clinical disease
types of hyperuricemia and gout; FIG. 19 is an explanatory diagram
showing haplotype of ABCG2; and FIG. 20 is an explanatory diagram
showing an onset risk by ABCG2 dysfunction in each type of
gout.
[0134] A haplotype of ABCG2 having a Q126X variant was named as
"*3". This is a haplotype whose function of ABCG2 becomes zero. A
haplotype of ABCG2 having a Q141K variant was named as "*2". This
is a haplotype whose function of ABCG2 becomes 1/2. A haplotype
having neither Q126X nor Q141 K variant was named as "*1". This is
a haplotype whose function of ABCG2 is normal.
[0135] FIG. 21 is a table showing frequency of ABCG2 function in
5005 Japanese individuals; FIG. 22 is an explanatory diagram
showing PAR % (Population attributable risk proportion percent) of
ABCG2 dysfunction for hyperuricemia; FIG. 23 is an explanatory
diagram showing the significant increase of serum uric acid level
by ABCG2 dysfunction; and FIG. 24 is a table showing effects of
ABCG2 dysfunction, BMI, alcohol comsumption, and the like, on the
serum uric acid levels.
[0136] Furthermore, the present inventors disclosed an ABCG2
protein function as a urate transporter in Patent Literature 5, and
they additionally have studied a urate excretion mechanism.
[0137] FIG. 25 is an explanatory diagram showing a transport
mechanism of urate; FIG. 26 is an explanatory diagram showing the
relationship between ABCG2 dysfunction and gout risk; and FIG. 27
is an explanatory diagram showing a structure and variations of
ABCG2 (Non-Patent Literatures 1 to 2, 8 and 10).
[0138] ABCG2 gene variants are observed in 80% of gout patients,
but in 50% of healthy individuals. All coding regions of ABCG2 gene
of 90 subjects having hyperuricemia were subjected to sequencing,
and only six variants with amino acid substitution were found.
Three of them show high frequency, and one of them did not cause
dysfunction of urate excretion (Non-Patent Literature 8). The most
important gene variations are Q126X and Q141K (Patent Literature
5). The Q126X variation was observed in 5.5% of Japanese, and this
results in no function. The Q141K variation was observed in as high
as 53.6% of Japanese, and this decreases the function to half. When
the Q141K variation is present, the amount of proteins to be
produced is the same as in the wild type, but transporter expressed
on the cell membrane becomes half, thus decreasing the function to
half. These two variations do not occur in one chromosome
concurrently. Accordingly, by checking whether one variation is
present or both variation are present, population risk can be
determined in a simple manner. However, if both are normal, there
are possibilities that other variants are present.
[0139] FIG. 28 is an explanatory diagram showing a relationship
between ABCG2 function and the onset risk of gout for each age
group (Non-Patent Literature 9).
[0140] As a result of examination of 705 gout patients, the onset
risk of gout is lowest in the fortieth and the onset risk is
2.3-fold in the group in which the ABCG2 function is 75% (i.e.,
3/4). The onset risk in the fiftieth is 2.5-fold in the group in
which ABCG2 function is 75%. In particular, the onset risk in the
twenties and younger is exceptionally high. The onset risk of gout
becomes as high as 22-fold in the twenties and younger in the group
in which the ABCG2 function is 25% (i.e., 1/4). In the group in
which the function is 25%, the onset risk is extremely high also in
the thirties and the fortieth.
[0141] In Meiji era, it is said that there was few gout patients in
Japan. In that era, when a gene variations are present, the serum
uric acid levels are properly increased. Since uric acid also has
an antioxidative effect, uric acid that is present at a proper
blood level has a good effect on the body. Today, however,
hypertrophication and lack of physical activity, together with the
risk of gout gene, which is originally present in the background of
Japanese, are a major factor of gout. Thus, the number of gout
patients is increasing.
[0142] FIG. 29 is an explanatory diagram showing disease type
classification of hyperuricemia (Non-Patent Literatures 6 and
10).
[0143] It was found that the ABCG2 transporter is expressed also in
the kidney and the intestinal tract. Conventionally, the
hyperuricemia is classified into two disease types, that is, an
"overproduction type" in which production of urate is increased, an
"underexcretion type" in which urate excretion from kidney is
decreased", and a "combined type" of the above-mentioned two types.
Decrease of excretion function due to the ABCG2 gene variations is
predicted to result in the "underexcretion type". However, it is
shown that an ABCG2 gene variations increase the amount of urate
excretion from the kidney (amount of urinary urate excretion) in
hyperuricemia cases. A high amount of the urinary urate excretion
is diagnosed to be the "overproduction type" or the "combined type"
in the conventional disease type classification. However, in an
example in which ABCG2 excretion functions decrease to 1/4 or less,
the "overproduction type" or the "combined type" reaches 90% of
hyperuricemia patients. On the other hand, it was shown that 2/3 of
urate was excreted from the kidney, and 1/3 of urate was excreted
from the intestinal tract.
[0144] However, it has been demonstrated, using ABCG2 knockout
mice, that when the urate excretion function of ABCG2 was
decreased, the serum uric acid levels and the urate excretion
amount from the kidney were increased, and on the contrary, the
urate excretion amount from the intestinal tract is significantly
decreased. Urate excretion to bile was not changed (Non-Patent
Literature 6).
[0145] From these results, the concept of "extra-renal
underexcretion type" hyperuricemia can be advocated as a new
disease type of hyperuricemia. Furthermore, the name of
"overproduction type" hyperuricemia in the conventional
classification including "extra-renal underexcretion type"
hyperuricemia and "genuine overproduction type" hyperuricemia can
be changed to the name "renal overload type" hyperuricemia.
[0146] In selection of drugs, for the "renal overload type" that is
the "conventional overproduction type", urate synthesis inhibitory
drugs are basically used, and for the "underexcretion type",
uricosuric agents are basically used. When the uricosuric agents
are used, or when urinary pH is acidic, it is considered that a
urine alkalizer may be used in combination from the viewpoint of
promoting urate excretion.
[0147] Obtaining information of the degree of the gout risk by an
ABCG2 genetic test enables early prevention and early intervention
of medical care, and start of medication to be considered by the
result of the test. According to the present guidelines, medicament
therapy is to be started when the serum uric acid level is 8.0
mg/dL, 9.0 mg/dL or more, but before the serum uric acid level
becomes such a high value, onset of gout occurs in some
individuals. In an ABCG2 gene high-risk group, it is necessary to
carry out early intervention to improvement of lifestyle habit and
early medication, if necessary.
[0148] Hyperuricemia may be involved in not only urate deposition
diseases (gout and renal disorders) but also cardiovascular
diseases. Thus, the present invention contributes to
self-prevention by individuals having high genetic risk, for
example, reducing body weight or taking care of diet or exercise,
for example, in a case where the individual is obese.
[0149] Furthermore, test of the combination of gene polymorphisms
enables estimation of clinical disease type and evaluation of
recommended treatment policy including drugs to be used to be
evaluated.
[0150] Furthermore, in Patent Literature 5, the present inventors
disclose the case where the variation of genes encoding ABCG2
protein include SNP of at least any one of Q126X, Q141K, G268R,
S441N, and F506SfsX. Herein, the present inventors have further
studied the ABCG2.
[0151] Wild-type human ABCG2 cDNA, which had been inserted into
pcDNA3.1(+) vector with a myc tag attached at the N-terminal, was
used (Non-Patent Literature 8). Variants of ABCG2 (F208S, P269S,
E334X, L447V, S486N, R575X, and C608X) were introduced by the
site-directed mutagenesis technique using the myc-ABCG2 wild
type/pcDNA3.1(+) vector as a template.
[0152] HEK293 cells were seeded into 12-well plate at
1.5.times.10.sup.5 cells/dish. After about 24 hours, the cells were
transfected with 0.5 g/dish of the wild type or each variant
myc-ABCG2/pcDNA3.1(+) vector. After the cells were cultured for 48
hours, they were collected using a RNA-solve reagent to obtain a
reverse transcription product. To perform a quantitative analysis
of mRNA, the real-time PCR reaction using the obtained reverse
transcription product and SYBR GreenER qPCR SuperMix Universal
(Invitrogen) was detected and analyzed with CHROMO4. The mRNA
amount of the ABCG2 wild type or each variant was normalized by the
mRNA amount of .beta. actin.
[0153] Expression analysis by the western blotting was carried out,
and the cells were immunostained to be visualized and observed by
using a confocal microscope. Furthermore, cell membrane vesicles
were prepared from HEK293 cells, and subjected to a transport
experiment.
[0154] FIG. 30 is an explanatory diagram showing positions of seven
kinds of amino acid variations of ABCG2. Among the ATP binding
sites, Walker A sequence, Walker B sequence, and signature C
sequence are shown by a rectangle, respectively. To red circles
showing positions of variation, indicators showing the name of
variants are added. "#" represents an N-type glycosylation binding
site (N596), and "*" represents cysteine residues for disulfide
bonds (C592, C603 and C608).
[0155] FIG. 31 shows quantitation of mRNA and protein of the wild
type and variant ABCG2 in HEK293 cells. FIG. 31(A) is a graph
showing comparison of the mRNA amount when wild type and variant of
ABCG2 are transiently introduced into HEK293 cells, FIG. 31(B) is a
western blotting photograph showing whole cell lysate, and FIG.
31(C) is a western blotting photograph showing crude membrane
separated from a polyacrylamide gel, transferred to a PVDF
membrane, then indicated with an anti-myc antibody, and detected by
chemiluminescence.
[0156] In order to analyze an effect of each variant of ABCG2 on
the expression amount, HEK293 into which myc-ABCG2 expression
vectors of wild type and each variant had been transiently
introduced, and in which quantitation PCR of mRNA was carried out.
As a result, no significant difference in mRNA expression amount
was observed between the wild type and each variant.
[0157] When western blotting was carried out using a whole cell
lysate and a crude product membrane, in wild-type ABCG2, a band was
observed in the position of about 80 kDa in the molecular weight.
In P269S, L447V, and S486N, a band was detected in the same
molecular weight as in the wide type, but the expression amount was
decreased to about 60% of that of the wild type in L447V and S486N.
On the other hand, in F208S, E334X, and R575X, although there is no
significant change in the RNA expression amount, normal expression
of protein was not observed. Furthermore, C608X produces a band
having a slightly higher molecular weight than that of the wild
type, the expression amount of protein was decreased to 20% or
less.
[0158] FIG. 32 shows confocal microphotographs showing
intracellular localization in the wild type and variant ABCG2 in
LLC-PK1 cells. The wild type and variant myc-ABCG2 were transiently
introduced into LLC-PK1 cells. The cells were stained using an
anti-myc antibody and TO-PRO3. Green shows ABCG2 and gray shows
nucleus.
[0159] In order to analyze an influence of each variation of ABCG2
on intracellular localization, the myc-ABCG2 expression vectors of
the wild type and each variant were transiently introduced into
LLC-PK1 cells, and localization patterns were compared. When the
cells were immunostained using an anti-myc antibody and observed by
a confocal microscope, it was shown that the wild-type ABCG2 was
localized on the apical membrane surface of the LLC-PK1 cells. The
results were consistent with the localization in a living body.
[0160] As a result of examination of the intracellular localization
of ABCG2 of each variant, in P269S, L447V, S486N, and C608X, ABCG2
was observed to be expressed on the apical membrane surface similar
to the wild type. On the other hand, in E334X and R575X, ABCG2 was
not observed to be expressed on the apical membrane, but observed
to be accumulated in the cell. In F208S, as in the western
blotting, a signal was not detected.
[0161] FIG. 33 is a western blotting photograph of protein
quantitation using a cell membrane vesicle expressing a wild type
and variant ABCG2. Cells membrane vesicle prepared by HEK293 cells
expressing a wild type and variant ABCG2 were isolated by
polyacrylamide gel, transferred to the PVDF membrane, labeled with
an anti-myc antibody, and detected by chemiluminescence. Band
density of the myc-ABCG2 in which normal expression was observed
was shown.
[0162] In order to analyze an influence of each variant of ABCG2 on
the urate transport activity, comparison of the transport activity
using a cell membrane vesicle was carried out. HEK293 cells into
which wild-type and variant myc-ABCG2 expression vectors had been
transiently introduced were harvested, and subjected to western
blotting using a cell membrane vesicle, the same results as in the
crude product membrane were observed. The ABCG2 expression amount
per protein in the variant expression vesicle was decreased to
1.16-fold as compared with that of wild type in P269S, 0.70-fold in
L447V, 0.58-fold in S486N, and 0.37-fold in C608X, and expression
of normal protein was not observed in F208S, E334X, and R575X.
[0163] FIG. 34(A) is a graph showing [.sup.14C] urate transport by
an ABCG2 variant, and FIG. 34(B) is a graph showing transport
activity normalized by ABCG2 protein.
[0164] A urate transport experiment was carried out using a cell
membrane vesicle prepared by HEK293 cells expressed by wild type
and variant ABCG2. As a result, while P269S maintains the same
level of urate transport activity as that of the wild type, no
urate transport was observed in F208S, E334X, and R575X. In 608X,
decrease of the transport ability with the decrease of the
expression amount was observed, but the urate transport activity
per expression amount of ABCG2 protein was the same level as that
of the wild type. Furthermore, in L447V and S486N, although the
intracellular localization is normal, the urate transport activity
is drastically decreased.
[0165] When one gene is a causative gene of a disease, it may be
considered that all variations of the gene are counted as a disease
risk factor, but in the case of ABCG2, for example, V12M and P269S
variations do not have an effect on the urate transport. Therefore,
it is not considered that these variations have an effect on the
onset risk of gout. In analysis of genes for the purpose of risk
prediction of the onset risk of hyperuricemia and gout, such
variations may not be considered as a factor that brings the risk
increase. On the other hand, since variations other than the two
variations have an effect on the urate transport function, similar
to Q126X and Q141K, it is considered that such variations increase
the onset risk of hyperuricemia and gout.
[0166] F208S expressed mRNA, but expression of protein was not
observed, and the urate transport function was not observed. Since
E334X and R575X are nonsense variations and do not express normal
protein, it is natural that they do not have a transport function.
However, although C608X is a nonsense variation, it maintained a
part of the transport function. This is thought to be partially
because C608X has termination codons after six transmembrane sites.
Furthermore, the western blotting band of C608X is shifted to
slightly higher molecular weight side as compared with the wild
type. This is suggested that a three-dimensional structure that is
different from the wild type is formed because C608 that is
suggested to be important for formation of the disulfide bond is
deleted.
[0167] According to the result of immunostaining in vitro, in L447V
and S486N, intracellular localization was observed at the brush
border membrane side similarly to the wild type, and the protein
expression amount was not so decreased as compared with that of the
wild type, but urate transport is hardly detected.
[0168] FIG. 35 is a table showing polymorphism and variation of
ABCG2, which were analyzed in the above-mentioned analysis and
analysis in Non-Patent Literature 6.
[0169] An effect of 13 types of variations and polymorphisms of
ABCG2, in which amino acid is substituted, on the urate transport
function was evaluated. R575X is already known (Non-Patent
Literature 11). However, it is the first time to analyze its
function by the present invention. F208S, P269S, and E334X were
analyzed in terms of function of the substrate such as methotrexate
(Non-Patent Literatures 11 to 14), resulting in that the change in
the urate transport activity seems to be the same as in the change
in the transport activity with respect to the other substrate.
L447V, S486N, and C608X are novel variations. These variations were
found as a result of analysis of specific population, that is,
population of human having hyperuricemia, gout, or the like, and
therefore, variations of ABCG2 gene are likely to be accumulated in
such population. The results reflect the strength of the
relationship between the ABCG2 gene and hyperuricemia, gout, or the
like.
[0170] The present inventors have further investigated NPT1.
[0171] FIG. 36 is a table showing the results of analysis of the
relationship between gout and a gene polymorphism rs1165196 of
NPT1/SLC17A1.
[0172] Gene polymorphism (SNP) (rs1165196, I269T) of NPT1/SLC17A1
was analyzed in 545 male gout patients and 1115 male subjects
having normal uric acid levels. As a result, in gout with renal
urate underexcretion (RUE gout) (FE.sub.UA of less than 5.5%), it
was found that the variation (rs1165196, I269T) of NPT1/SLC17A1
significantly decreases the risk of gout. Odds ratio was 0.73-fold
(95% confidence interval: 0.54 to 0.97, P-value: 0.031).
[0173] Furthermore, the immunohistochemical analysis shows that
NPT1 is expressed at the apical side of the proximal tubules of
human kidney. Furthermore, in also function analysis using living
cells in which NPT1 was expressed in Xenopus laevis oocytes, it was
found that I269T enhanced the urate excretion function as compared
with the wild type. Thus, it was shown that I269T was a
gain-of-function type variation, promotes the urate excretion by
NPT1, and supports to decreases the-risk of gout.
[0174] From this analysis, in all the gout (All gout) and gout in
which renal urate underexcretion is not observed (Non-RUE gout)
(FE.sub.UA: 5.5% or more), significant differences were not
observed in the onset risk of gout. However, it was found that in
the other analysis with number of samples increased, a gene
polymorphism (SNP) (rs1165196, I269T) of NPT1/SLC17A1 also
significantly decreases the risk of all the gout (All gout).
[0175] FIG. 37 is a photograph showing localization of NPT1 in
human kidney by immunohistochemistry.
[0176] With an anti-human NPT1 antibody (SANTA CRUZ, Santa Cruz,
Calif., USA) (diluted at 1:500) produced from rabbit, tissue
sections of human kidney were incubated overnight at 4.degree. C.
and treated with an anti-rabbit peroxidase-labelled polymer
(Envision+: Dako, Tokyo, Japan) for 30 min, and immunoreactions
were detected by staining with diaminobenzidine (0.8 mM). A bar in
the photograph is 50 .mu.m.
[0177] As a result, it was clarified that NPT1 was localized at the
apical side in the proximal tubules in the human kidney.
[0178] FIG. 38 is a diagram showing a physiological function of
NPT1.
[0179] NPT1 mediates the excretion of urate into urine at the
apical side of the proximal tubules in human kidney, and acts to
decrease the serum uric acid level so as to play an important
physiological role in regulation of serum uric acid levels.
[0180] Furthermore, I269T that is a gain-of-function type
(gain-of-function type) variation of NPT1 acts so as to promote
urate excretion into urine (resulting in increasing FE.sub.UA), and
decreases the serum uric acid levels. Therefore, it was
pathologically clarified that the variation significantly decreases
the risk of gout.
[0181] Furthermore, QTL analysis of serum uric acid levels for
rs56027330 (G279R) of NPT4/SLC17A3 in 5017 male and female Japanese
individuals was carried out. As a result of correction based on
sex, BMI, ABCG2 function, and NPT1/SLC17A1 (rs1165196; I269T),
G279R of NPT4 showed significant (P=0.03) relationship with respect
to the serum uric acid levels. Thus, the relationship between the
rare gene polymorphism of NPT4 and the serum uric acid levels was
demonstrated for the first time, and it was suggested that NPT4 was
likely to be associated with uric acid-related diseases including
gout and hyperuricemia or inflammatory diseases related
thereto.
[0182] FIG. 39 shows a graph and a western blotting photograph
showing results of urate transport analysis of mutated ABCG2.
[0183] In order to clarify an effect of the urate transport
activity on the ABCG2 function, using membrane vesicles expressing
the wild type and variant ABCG2 protein, urate transport activities
of seven types of variants were examined. ATP-dependent urate
transport was remarkably decreased in V178I, N299S, E311K, V508I,
and A634V, and was nearly eliminated in G462R and V516M. Western
blot analysis showed that the expression amount of ABCG2 protein on
the membrane vesicles was not so different among V178I, N299S, E311
K, V508I, V516M, and A634V, but was remarkably decreased in
G462R.
[0184] FIG. 40(A) is a graph showing the result of F489L (exon 12),
one of nonsynonymous variants of ABCG2 gene, found in the sequence
analysis of gout cases, and FIG. 40(B) is a graph showing the
result of D620G (exon 16), one of-nonsynonymous variants of ABCG2
gene, found in the sequence analysis of gout cases.
[0185] In order to examine the nonsynonymous variants of ABCG2
gene, in 500 gout cases, the exon of ABCG2 gene was sequenced. As a
result, as the other nonsynonymous mutation, F489L (exon 12) and
D620G (exon 16) were identified.
[0186] FIG. 41(A) is a table showing the results of analysis of the
association between hyperuricemia and URAT1 nonsynonymous variants;
and FIG. 41(B) is a table showing the results of analysis (with
adjustment by Q126X and Q141K variations) of the relationship
between hyperuricemia and URAT1 nonsynonymous variants.
[0187] The subjects include 2209 male patients with hyperuricemia
and 1388 controls. A significant relationship between rare
polymorphisms W258X and R90H of URAT1 gene (urate reabsorption
transporter gene) and gout was observed.
[0188] FIG. 42 is a table showing the results of genome-wide
association study of gout followed by replication analysis using a
custom chip.
[0189] When a meta-analysis was carried out based on the analysis
results of the primary analysis (GWAS, 945 clinically diagnosed
gout cases, and 1213 controls) and the secondary analysis
(replication study using a custom chip, 1048 clinically diagnosed
gout and 1334 controls), a significant relationship was observed in
a gene polymorphism (SNP) (rs2285340) of NRXN2-SLC22A12/URAT1, a
gene polymorphism (SNP) (rs 1165196) of SLC17A1/NPT1, a gene
polymorphism (SNP) (rs11758351) of HIST1H2BF/HIST1H4E, and a gene
polymorphism (SNP) (rs4496782) of HIST1H2BE/HIST1H4D. Furthermore,
other than the above, FAM35A (rs7903456, Chromosome 10) showed a
significant relationship with renal underexcretion gout (RUE gout)
by the replication analysis.
[0190] FIG. 43 is a table showing the results of analysis of the
change of urinary coproporphyrin based on the function of
ABCG2.
[0191] The subjects include 509 examinees of health examination.
Urinary coproporphyrin as one type of a porphyrin body was analyzed
by a value corrected using urine creatinine (.mu.g/gCrea). ABCG2 is
known to transport not only urate but also porphyrin, and ABCG2 was
found to be associated with porphyrin. Furthermore, when cases of
Q126X homozygote of ABCG2 (ABCG2 function: 0%) was analyzed,
urinary coproporphyrin was 16 .mu.g/gCrea. The result was
consistent with the results shown in FIG. 43. Furthermore,
protoporphyrin in whole blood was 92.5 .mu.g/dl (normal value: 40
.mu.g/dl or less), and apparently increased from the normal value.
These findings show that ABCG2 dysfunction increases porphyrin in
human cells, and suggest that it is associated with pathologic
conditions such as photosensitivity.
[0192] FIG. 44 is a table showing the results of analysis of the
relationship between the function of ABCG2 and cerebral stroke.
[0193] A significant association was observed between the gene
polymorphisms of ABCG2 and cerebral stroke as an inflammatory
disease.
[0194] Furthermore, ABCG2 may be involved in inflammatory diseases
via the effect on high-capacity urate transport of urate or the
like. Also in atrial fibrillation, a kind of arrhythmia, it is
reported that inflammation is involved in its pathologic conditions
(Non-Patent Literatures 15 to 16). According to the analysis by the
present inventors, 20 subjects having previous atrial fibrillation
were extracted from 4999 subjects of health examination, the
distribution of the ABCG2 functions in the 20 subjects showed
significant difference (P=0.01) from that of the subjects not with
atrial fibrillation. The analysis suggests that ABCG2 dysfunction
is related to pathologic conditions of atrial fibrillation.
[0195] FIG. 45 is a table showing the results of analysis of the
serum uric acid levels in ulcerative colitis cases based on the
function of ABCG2.
[0196] In ulcerative colitis cases, ABCG2 dysfunction tends to
increase serum uric acid levels (SUA).
[0197] FIG. 46 is a table showing the results of analysis of the
serum uric acid levels before treatment in viral enteritis cases
based on the function of ABCG2.
[0198] A significant association was observed between the ABCG2
function in patients with viral enteritis disease as viral
intestinal disease and the increase in the uric acid levels before
treatment. Furthermore, the uric acid levels before treatment
significantly increased as the ABCG2 function decreased. An average
value of the convalescent serum uric acid levels was 4.85.+-.0.26
mg/dl, and remarkably increased before treatment. Subjects include
58 subjects with pediatric viral enteritis (30 male subjects and 28
female subjects).
[0199] FIG. 47(A) is a table showing the results of analysis based
on the ABCG2 function and the age at which hemodialysis is
introduced in hemodialysis cases. FIG. 47(B) is a table showing the
results of analysis of the ABCG2 function and the serum uric acid
levels in hemodialysis cases.
[0200] When the relationship between the ages at which hemodialysis
is introduced and the ABCG2 function with respect to subjects
including 139 hemodialysis cases (101 male subjects and 38 female
subjects), it was found that a variations of ABCG2 made the ages at
which hemodialysis is introduced earlier. Furthermore, when the
relationship between the serum uric acid levels and the ABCG2
function was examined in 106 cases (73 male subjects and 33 female
subjects) without oral administration of therapeutic agents for
gout and hyperuricemia, it was found that the ABCG2 variations
increased the serum uric acid levels extremely significantly.
[0201] FIG. 48(A) is a table showing the results of analysis of the
relationship between the onset age of gout and the ABCG2 function.
FIG. 48(B) is a table showing the results of analysis of the
relationship between the onset age of Parkinson's disease and the
ABCG2 function in patients with Parkinson's disease. FIG. 49 is an
explanatory diagram showing different influences of ABCG2
dysfunctions in gout and Parkinson's disease.
[0202] When Q141K variation of the ABCG2 gene was examined in 507
male gout cases, a significant association was observed between the
onset age of gout and the ABCG2 function. Furthermore, when Q141K
variation of the ABCG2 gene was examined in 1015 Parkinson's
disease cases as neurodegenerative diseases, a
significant-association was observed between the onset age of
Parkinson's disease and the ABCG2 function. Parkinson's disease and
ABCG2 polymorphism show inverse association, but the uric acid
levels needs to be controlled appropriately for prevention of
neurodegenerative diseases including Parkinson's disease.
[0203] Based on the above-mentioned examples and findings, a
molecule associated with the onset of gout of the present invention
includes any one protein or cDNA of CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, ABCG2, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D
and FAM35A, or a combination thereof with any one protein or cDNA
of GLUT9, NPT1, URAT1 and NXRN2, and is capable of relating to the
onset of gout; or includes protein or cDNA of an ABCG2 variant, and
is capable of selectively and ATP-dependently decreasing excretion
of urate.
[0204] The present inventors similarly disclose a urate transporter
formed of protein having ABCG2 and is capable of selectively and
ATP-dependently exporting uric acid as a urate transporter as a
molecule associated with the onset of gout, in Patent Literature 5;
and also disclose a urate transporter formed of protein including
SLC2A9/GLUT9, and is capable of selectively and ATP-dependently
exporting uric acid, in Non-Patent Literature 2.
[0205] Based on them, any one of CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and
FAM35A may be combined with ABCG2, SLC2A9/GLUT9, GLUT9, NPT1,
URAT1, and NXRN2.
[0206] A method for evaluating a uric acid-related disease
diathesis and an inflammation-related disease diathesis of the
present invention includes evaluating whether or not a subject has
a diathesis capable of inducing urate regulation failure, or a
state or a disease attributable to the failure. The evaluating
includes a step of detecting a variation of a gene encoding at
least any one protein selected from CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D and
FAM35A, using a test sample containing human genes of the
subject.
[0207] For detection of a variation of a gene encoding any one
protein of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, detection of an
SNP or a gene polymorphism having a relationship of linkage
disequilibrium with the SNP may be used.
[0208] Similar to the above, CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR,
MAP3K11, NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A
may be combined with ABCG2, SLC2A9/GLUT9, GLUT9, NPT1, URAT1, and
NXRN2.
[0209] Note here that CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, as well
as ABCG2, SLC2A9/GLUT9, NPT1, URAT1, and NXRN2 genes include cDNAs
derived from human, homogeneous genes derived from human which
hybridize with a DNA consisting of a complementary base sequence
under a stringent condition and which encode a polypeptide having a
urate transport capability, and homologues thereof in mammals.
[0210] Examples of the uric acid-related disease and the
inflammation-related disease include hyperuricemia, gout,
rheumatoid arthritis, osteoarthritis, infertility, cerebral stroke,
neurodegenerative disease, ischemic heart disease, chronic kidney
disease, renal dysfunction, urolithiasis, kidney stone, aneurysm,
arrhythmia including atrial fibrillation, inflammatory bowel
disease, enteritis, functional dyspepsia, viral intestinal disease,
and photosensitivity.
[0211] Furthermore, a higher serum uric acid level is apt to
develop uric acid-related diseases and inflammation-related
diseases. Accordingly, when the level is equal to or more than a
predetermined level such as, for example, 8.0 mg/dl, it may be
evaluated that a subject has a high diathesis capable of inducing
urate regulation failure or a state or a disease attributable to
the failure. The threshold value can be appropriately changed, for
example, to 7 or 9.
[0212] A method for evaluating a uric acid-related disease
diathesis and an inflammation-related disease diathesis of the
present invention is a method for evaluating whether or not a
subject has a diathesis capable of inducing urate regulation
failure, or a state or a disease attributable to the failure. The
evaluating includes a step of detecting a variation of a gene
encoding ABCG2 protein using a test sample including human genes of
the subject. Detection of the variation of the gene is detection of
an SNP or a gene polymorphism having a relationship of linkage
disequilibrium with the SNP. When a subject has a SNP that
generates an amino acid variation of at least any one of R113X,
F208S, L447V, S486N, R575X, C608X, P269S, E334X, F489L, and D620G,
the method may evaluate that the subject has a diathesis capable of
inducing urate regulation failure, or a state or a disease
attributable to the failure.
[0213] The present inventors disclose, in Patent Literature 5,
Q126X, Q141K, G268R, S441 N, and F506SfsX as the similar ABCG2 gene
variations. In particular, the present inventors disclose the
similar method, when Q126X alone or combination of Q126X and Q141K
have SNP.
[0214] Based on the disclosures, any one of R113X, F208S, L447V,
S486N, R575X, C608X, P269S, E334X, F489L, and D620G may be combined
with Q126X, Q141K, G268R, S441N, and F506SfsX.
[0215] Evaluation can be carried out as follows based on Q126X and
Q141K as shown in, for example, FIG. 26.
[0216] When a gene encoding Q of Q126X is C/C and a gene encoding Q
of Q141K is C/C, the function of ABCG2 is evaluated to be normal;
when a gene encoding Q of Q126X is C/C, and a gene encoding Q of
Q141K is A/C, the function of ABCG2 is evaluated to be 3/4; when a
gene encoding Q of Q126X is T/C and a gene encoding Q of Q141K is
C/C, the function of ABCG2 is evaluated to be 1/2; when a gene
encoding Q of Q126X is C/C and a gene encoding Q of Q141K is A/A,
the function of ABCG2 is evaluated to be 1/2; when a gene encoding
Q of Q126X is T/C and a gene encoding Q of Q141K is A/C, the
function of ABCG2 is evaluated to be 1/4; and when a gene encoding
Q of Q126X is T/T and a gene encoding Q of Q141 K is C/C, ABCG2 is
evaluated to have no function. The method evaluates that a
diathesis capable of inducing urate regulation failure, or a state
or a disease attributable to the failure is evaluated to be high
depending on a degree of loss of the function of ABCG2. Note here
that C/C and the like means "derived from mother side/derived from
father side".
[0217] The ABCG2 dysfunction includes determining the equivalent
degree of BMI reduction, an amount of absolute alcohol intake, age,
and sex, which correspond to the effect on the serum uric acid
levels, 1/4 decrease of the ABCG2 function corresponds to the BMI
increase of about 1.97 or about 552 ml per week of absolute alcohol
intake. Use of this indicator contributes to not only prevention of
onset of diseases such as gout, but also health case by the
consciousness about lifestyle habit.
[0218] That is to say, it can be evaluated that an effect of 1/4
decrease of the ABCG2 function on the serum uric acid levels
corresponds to the increase of about 0.193 mg/dl (95% confidence
interval: 0.150-0.235 mg/dl), the increase of BMI of 1 kg/m.sup.2
corresponds to the increase of the serum uric acid level of about
0.098 mg/dl (95%confidence interval 0.087-0.108 mg/dl), and about 1
g per week of absolute alcohol intake corresponds to the increase
of the serum uric acid level of about 0.00035 mg/dl (95% confidence
interval 0.00017-0.00053 mg/dl).
[0219] Determination of gene polymorphisms can be carried out using
human blood or tissues as a material, by a direct sequencing
method, a BAC array CGH method, a FISH method, an RFLP method, a
PCR-SSCP method, an allele-specific oligonucleotide hybridization
method, a TaqMan PCR method, an invader method, an HRM method, a
MALDI-TOF/MS method, a molecular beacon method, an RCA method, a
UCAN method, a nucleic acid hybridization method using a DNA chip
or a DNA microarray, and the like.
[0220] SNPs can be detected directly from a genomic DNA by a direct
sequencing method and the like.
[0221] Also, a particular genomic DNA region may be amplified using
a clone, or a PCR method, an LCR method, an SDA method, an RCK
method, a LAMP method, an NASBA method and the like, and then,
determination of a base sequence of a portion of an allele
containing at least a polymorphic site, detection by a probe
specifically hybridizing with a polymorphic site, and measurement
of a molecular weight of a gene fragment containing a polymorphic
site may be performed.
[0222] SNPs of an amplified product can be determined by
determination of the base sequence, measurement of the molecular
weight by a MALDI-TOF mass analysis, and the like, analysis of the
restriction enzyme fragment length, detection by SSCP,
electrophoresis, and the like.
[0223] For example, the TaqMan method is a method in which a
hybridization of an allele-specific oligonucleotide with a template
is carried out concomitantly with a PCR method, and SNPs are
detected using a fluorescence energy transfer phenomenon. When an
allele-specific probe labeled with a fluorescent dye and a quencher
is hybridized with a target site and PCR is carried out using a
primer designed to amplify a region including that site, the
hybridized probe is cleaved by a 5' nuclease activity of Taq
polymerase, concomitantly with the progress of an extension
reaction from the primer. Separation of the fluorescent dye and the
quencher yields fluorescence, and amplification of the template by
the PCR reaction exponentially enhances a fluorescence intensity.
When two allele-specific probes are labeled with different
fluorescent dyes, a homozygote and a heterozygote can be
distinguished from each other in one assay.
[0224] The invader method is a method using two oligonucleotide
probes, and is based on an enzyme reaction which recognizes and
cleaves a specific structure formed by these probes and a template
DNA. A target base sequence is recognized by two different probes,
i.e., an invader probe substantially complementary to a first site
of the target base sequence, and an allele probe which, on its
3'-terminal side, is substantially complementary to a second site
of the target base sequence and which, on its 5'-terminal side,
contains a flap not complementary to the template and forming a
single strand. When these probes hybridize with adjacent regions of
the template, the 3'-terminus of the invader probe invades an SNP
site, and the structure is cleaved by an enzyme to release the
flap. By labeling the flap in advance, it is possible to quantify
the flap released. By preparing two sets of flap-FRET probes and
labeling them by different fluorescent dyes, a homozygote and a
heterozygote can be distinguished from each other in one assay.
[0225] The MALDI-TOF mass analysis is a method in which a primer
adjacent to an SNP site is prepared, a primer extension reaction of
only one base is carried out using a PCR-amplified test sample DNA
as a template and using ddNTP, and the ddNTP added is identified by
a mass analysis of extension reaction products. The method does not
need any fluorescent label of the primer, and can treat a large
number of test samples in a short time.
[0226] The RCA method is a method for applying a DNA-amplifying
means, in which a DNA polymerase moves on the template and
synthesizes a long complementary DNA using a circular
single-stranded DNA as a template, to SNP typing. Identification of
an SNP is carried out by the presence or absence of amplification
via the RCA method. That is to say, a single-stranded probe, which
can anneal with a genomic DNA and can become circular, is
hybridized with a genomic DNA to carry out the chain reaction. When
the terminus of the probe is set to an SNP site to be identified,
matching of the site leads to amplification via RCA because of
linkage and circularization, but mismatching does not lead to RCA
amplification because of no linkage and no circularization. The SNP
can be determined by identification of these two amplification
reactions.
[0227] The DNA chip method is a method for carrying out
hybridization with a PCR-amplified, fluorescence-labeled cDNA or
cRNA using a DNA chip prepared by arranging oligonucleotide probes
containing a polymorphic site on a microarray. The method can
detect many SNPs rapidly.
[0228] Examples of methods for determining polymorphisms in an
amino acid sequence include a proteome analysis by a
two-dimensional electrophoresis method or a microfluidics method,
peptide mapping and an amino acid sequence analysis using a mass
spectroscope, an amino acid sequence analysis by a protein
sequencer, a method for detecting the interaction between a
polypeptide and a ligand using a protein chip and the like.
[0229] For example, the two-dimensional electrophoresis method
usually conducts isoelectric point electrophoresis for the first
dimension and SDS-PAGE for the second dimension, and can separate
several thousand proteins on one plate of gel. For the isoelectric
point electrophoresis, an amphoteric carrier or an immobilized pH
gradient gel strip is used. For the SDS-PAGE, a continuous buffer
solution system using one buffer solution having a certain pH or a
discontinuous buffer solution system using multiple buffer
solutions having a different pH is used. It is also possible to use
a low BIS concentration gel electrophoresis, a concentration
gradient gel electrophoresis, tricine-SDS-PAGE and the like,
depending on the type of proteins to be separated. The proteins
separated can be detected using Coomassie Blue staining or silver
staining or using a fluorescent reagent on the gel with high
sensitivity. A western blotting method using an antibody against an
ABCG2 polypeptide can be also used.
[0230] The MALDI-TOF/MS method which is one of mass analysis
methods is a method in which a protein test sample is mixed with a
matrix absorbing a laser beam such as sinapic acid, the mixture is
dried and then irradiated with a high-energy pulse laser beam, the
protein test sample is ionized by energy transfer from the matrix,
and a molecular weight of the ion is analyzed on the basis of the
difference in flight time of a molecular ion of the test sample by
an initial acceleration. In order to fragmentize a peptide in the
inside of a mass spectrometer and to obtain an amino acid sequence,
an amino acid composition or the like by mass analysis of a
fragment, a tandem mass spectrometry in which multiple mass
separation portions are linked to each other is used. A triple
quadrupole type, a hybrid type, or an ion trap type analyzer using
an electrospray ionization method, and other analyzers are also
used.
[0231] The protein chip method can carry out comprehensively and
rapidly the interaction of a test sample with proteins, peptides,
antibodies, expressed proteins, and the like, which are arranged on
a basal plate.
[0232] The evaluation kit for a uric acid-related disease diathesis
and an inflammation-related disease diathesis according to the
present invention is a kit for evaluating whether or not a subject
has a diathesis capable of inducing urate regulation failure, or a
state or a disease attributable to the failure. The kit has means
for detecting at least any one SNP in CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A genes, or a gene polymorphism having
a relationship of linkage disequilibrium with the SNP, or a gene
polymorphism having a frequency of 1% or less, or a combination
thereof with a gene polymorphism including ABCG2, GLUT9, NPT1,
URAT1, and NXRN2, using a test sample containing human genes of the
subject.
[0233] Similar to the above, CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR,
MAP3K11, NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A
may be combined with ABCG2, SLC2A9/GLUT9, NPT1, URAT1, and
NXRN2.
[0234] As the SNPs of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, GLUT9, NPT1, URAT1, NXRN2, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A, rs4073582, rs671, rs2188380,
rs1260326, rs10791821, rs56027330, rs3775948, rs1165196, rs505802,
rs2285340 or rs506338, rs11758351, rs4496782, and rs7903456 can be
used, respectively.
[0235] As to the ABCG2 gene, means for detecting a SNP of at least
any one of R113X, F208S, L447V, S486N, R575X, C608X, P269S, E334X,
V178I, N299S, E311K, G462R, V508I, V516M, A634V, F489L, and D620G,
or a gene polymorphism having a relationship of linkage
disequilibrium with the SNP is provided.
[0236] That is to say, a polynucleotide including an ABCG2 gene
polymorphism, or a primer pair for amplifying a polynucleotide
containing a polymorphism of the ABCG2 gene or a DNA fragment
containing a polymorphism, or a polynucleotide for detecting a
polymorphism may be provided.
[0237] Similar to the above, R113X, F208S, L447V, S486N, R575X,
C608X, P269S, E334X, V178I, N299S, E311K, G462R, V508I, V516M,
A634V, F489L, and D620G may be combined with V12M, Q126X, Q141K,
P269S, S441N, and 506SfsX.
[0238] Examples of polynucleotides include both polyribonucleotides
and polydeoxyribonucleotides. They may be unmodified RNAs or DNAs,
modified RNAs or DNAs, and include, for example, DNAs, cDNAs,
genomic DNAs, mRNAs, unprocessed RNAs, their fragments and the
like.
[0239] Furthermore, polypeptides are those in which two or more
amino acids are linked by a peptide bond, and include relatively
short chain polypeptides referred to as peptides or oligopeptides,
and also long chain polypeptides referred to as proteins. The
polypeptides may contain amino acids other than 20 amino acids
encoded genetically, and modified amino acids. The modification
includes acetylation, acylation, ADP-ribosylation, amidation,
biotinylation, a covalent bond with lipids and lipid derivatives,
formation of a cross-linking bond, a disulfide bond, addition of a
sugar chain, addition of a GPI anchor, phosphorylation, prenylation
and the like in a main chain of peptide bonds, a side chain of
amino acids, an amino-terminus, and a carboxyl-terminus.
[0240] The inspection object of the present invention is a nonhuman
animal having a deficiency or overexpressing of at least any one
gene selected from CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11,
NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A genes, or
combination thereof with ABCG2, SLC2A9/GLUT9, NPT1, URAT1, and
NXRN2 genes with the above-mentioned gene, and the inspection
object may be provided with means for examining the urate transport
kinetics.
[0241] Nonhuman animals include, for example, mammals such as
mouse, and also include tissues and cells constituting their body.
Also, test samples are those containing polynucleotides derived
from organisms, and include body fluid, skin, hair root, mucosal
membrane, internal organs, placenta, cord blood, and the like,
collected from tissues and cells.
[0242] Similarly, nonhuman animals overexpressing a human ABCG2
gene or a nonhuman ABCG2 gene including at least any one variation
(in particular, gene polymorphism) of R113X, F208S, L447V, S486N,
R575X, C608X, P269S, E334X, V178I, N299S, E311K, G462R, V508I,
V516M, A634V, F489L, and D620G of a human ABCG2 gene or a nonhuman
ABCG2 gene; nonhuman cell lines or human cell lines having
deficiency of at least any one gene of CNIH2-PACS1, ALDH2,
MYL2-CUX2, GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A; nonhuman cell lines or human cell
lines overexpressing at least any one gene of human CNIH2-PACS1,
ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A or gene of at least any one gene of
nonhuman CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A; nonhuman cell
lines or human cell lines overexpression of a human ABCG2 gene or a
nonhuman ABCG2 gene including at least any of one variation of
R113X, F208S, L447V, S486N, R575X, C608X, P269S, E334X, V178I,
N299S, E311K, G462R, V508I, V516M, A634V, F489L, and D620G, or a
cell membrane vesicle prepared by such cell lines may be used.
[0243] Similar to the above, R113X, F208S, L447V, S486N, R575X,
C608X, P269S, E334X, V178I, N299S, E311K, G462R, V508I, V516M,
A634V, F489L, and D620G may be combined with V12M, Q126X, Q141K,
G268R, S441N, and F506SfsX.
[0244] Drugs for uric acid-related diseases and the drug for
inflammation-related diseases are drugs for reducing a diathesis
capable of inducing urate regulation failure, or a state or a
disease attributable to the failure, and contains a polynucleotide
encoding at least any one protein of CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and
FAM35A in the form capable of introducing it into cells or a
polypeptide corresponding to at least any one protein of
CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A in the form
capable of introducing it into cells. The former drug can stably
improve the urate transport for a long period, and the latter drug
can easily improve the urate transport by administration via
injection and the like.
[0245] Similar to the above mention, CNIH2-PACS1, ALDH2, MYL2-CUX2,
GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and
FAM35A may be combined with ABCG2, SLC2A9/GLUT9, NPT1, URAT1, and
NXRN2.
[0246] Note here that the form capable of introducing a
polynucleotide into cells means a form allowing introduction of
polynucleotide into cells and expression of any of CNIH2-PACS1,
ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4, HIST1H2BF/HIST1H4E,
HIST1H2BE/HIST1H4D, and FAM35A encoded so that any of intracellular
CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A genes express at
least any of CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A, respectively.
Similarly, the form capable of introducing a polypeptide into cells
means a form allowing introduction of the polypeptide into cells
and exertion of a function similar to that of at least any of
intracellular CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A.
[0247] CNIH2-PACS1, ALDH2, MYL2-CUX2, GCKR, MAP3K11, NPT4,
HIST1H2BF/HIST1H4E, HIST1H2BE/HIST1H4D, and FAM35A polynucleotides
can be obtained by a method of screening an existing cDNA library
using an oligonucleotide probe prepared on the basis of a known
nucleotide sequence, or a method such as RT-PCR using an
oligonucleotide primer.
[0248] ABCG2 not having any SNP in R113X, F208S, L447V, S486N,
R575X, C608X, P269S, E334X, V178I, N299S, E311K, G462R, V508I,
V516M, A634V, F489L, and D620G, and ABCG2 not having at least an
SNP in Q126X are preferred. In order to obtain a form capable of
introducing the ABCG2 polynucleotide into cells, for example, a
method using the polynucleotide as a bare DNA, or a method
formulating the polynucleotide in a form of a recombinant virus
vector is used. Virus vectors include those derived from genomes of
viruses belonging to Baculoviridae, Parvoviridae, Picornoviridae,
Herpesviridae, Poxyiridae, Adenoviridae, Picornaviridae and the
like.
[0249] Note here that as mentioned above, R113X, F208S, L447V,
S486N, R575X, C608X, P269S, E334X, V178I, N299S, E311K, G462R,
V508I, V516M, A634V, F489L, and D620G may be combined with V12M,
Q126X, Q141K, G268R, S441N, and F506SfsX.
[0250] Also, a polynucleotide expression vector may be introduced
into tissues or cells removed from a living body, and then, the
tissues or cells may be returned to the living body. In such a
case, a method can be used in which an expression vector
integrating a polynucleotide is introduced into cells by
transfection such as, for example, a microinjection method or an
electroporation method.
[0251] The polynucleotide in a virus vector or an expression vector
may be linked under a control of a promoter inducing systemic or
tissue-specific expression. When a virus vector is infected in a
kidney-specific manner, it is possible to introduce a recombinant
vector by percutaneously inserting a catheter into an artery and
then inserting the catheter into a renal artery while checking the
location of the catheter by X-rays.
[0252] A polypeptide such as ABCG2 can be produced by a genetic
engineering technique using the above-mentioned polynucleotide such
as ABCG2. That is to say, the polypeptide such as ABCG2 can be
obtained in vitro by preparing an RNA by an in vitro transcription
from a vector containing the polynucleotide, and carrying out an in
vitro translation using it as a template. When the polynucleotide
is recombined into an expression vector, it is also possible to
obtain the polypeptide such as ABCG2 as an expression product from
prokaryotic cells such as Escherichia coli or Bacillus subtilis,
from yeast, or from eukaryotic cells such as insect cells or mammal
cells.
[0253] Also, the polypeptide such as ABCG2 can be synthesized
according to a known chemical synthesis method.
[0254] The polypeptide such as ABCG2 may be provided as a peptide
derivative. Such a derivative contains a modification for
accelerating synthesis and purification, a modification for
accelerating physical and chemical stabilization, an activation
modification such as stabilization and instabilization or
conditioning for in vivo metabolism, and the like.
[0255] Other modifications in peptide derivatives include
acetylation, acylation, ADP-ribosylation, amidation, a covalent
bond of flavin, a covalent bond of a heme moiety, a covalent bond
of nucleotides or nucleotide derivatives, a covalent bond of lipids
or lipid derivatives, a covalent bond of phosphatidylinositol,
cross-linking, cyclization, a disulfide bond, demethylation,
formation of a cross-linking covalent bond, cystine formation,
pyroglutamate formation, formylation, gamma-carboxylation,
glycosylation, GPI-anchor formation, hydroxylation, iodination,
methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation, prenylation, racemization, a lipid bond,
sulfation, selenoylation and the like.
[0256] Specifically, the peptide derivatives can be prepared in the
form of a functional group produced as a side chain or as an
N-terminal group or a C-terminal group, in the range not destroying
any activity of a polypeptide such as ABCG2 and not giving any
toxicity to a composition containing the polypeptide. Examples
thereof include derivatives containing a polyethylene glycol side
chain which extends retainment of a polypeptide in the body fluid,
aliphatic esters of a carboxyl group, amides of a carboxyl group by
a reaction with ammonia or an amine, N-acyl derivatives of a free
amino group on an amino acid residue formed with an acyl moiety,
O-acyl derivatives of a free hydroxyl group formed with an acyl
moiety and the like.
[0257] The polypeptide such as ABCG2 also may be provided in the
form of a pharmaceutically acceptable salt. Such a salt includes
both a salt of a carboxyl group and an acid addition salt of an
amino group on the polypeptide.
[0258] Examples of salts of a carboxyl group include inorganic
salts such as a sodium, calcium, ammonium, iron, or zinc salt, as
well as salts with an organic base formed using an amine such as
triethanolamine, arginine, lysine, piperidine, and procaine.
Examples of salts of acid addition salts include salts with a
mineral acid such as hydrochloric acid or sulfuric acid, as well as
salts with an organic acid such as acetic acid or oxalic acid.
[0259] In order to formulate a polypeptide such as ABCG2 in the
form capable of introducing it into cells, for example, a fused
polypeptide in which a transmembrane peptide is linked to an
N-terminal side of the polypeptide is used. As the transmembrane
peptide, PTD of HIV-1 TAT or PTD of drosophila homeobox protein
Antennapedia can be used. The fused polypeptide can be prepared by
a genetic engineering technique, for example, using a fused
polynucleotide prepared by linking a polynucleotide such as ABCG2
and a PTD polynucleotide. It is also possible to prepare a fused
polypeptide linked with a transmembrane peptide by a method for
linking a polypeptide and a PTD peptide through a cross-linking
agent such as EDC or .beta.-alanine. Such a fused polypeptide can
be introduced by percutaneously inserting a catheter into an artery
and then inserting the catheter into a renal artery while checking
the location of the catheter by X-rays to introduce a recombinant
vector.
INDUSTRIAL APPLICABILITY
[0260] The present invention effectively evaluates whether or not a
subject has a diathesis capable of inducing urate regulation
failure, or a state or a uric acid-related disease and an
inflammation-related disease attributable to the failure, and
therefore contributes to prevention and early treatment of various
diseases related to abnormality in the uric acid levels, or uric
acid-related genes or gout-related genes. Furthermore, the present
invention contributes to treatment of uric acid-related diseases
without causing other undesirable effects even after the onset.
Accordingly, the present invention is effective to
inflammation-related diseases such as hyperuricemia, gout,
rheumatoid arthritis, osteoarthritis, infertility, cerebral stroke,
neurodegenerative disease, ischemic heart disease, chronic kidney
disease, renal dysfunction, urolithiasis, kidney stone, aneurysm,
arrhythmia including atrial fibrillation, inflammatory bowel
disease, enteritis, functional dyspepsia, viral intestinal disease,
and photosensitivity, and also effective to hypertension, obesity,
diabetes, a coronary artery disease, a cerebrovascular disease, a
kidney disease and the like which are likely to develop as a result
of complications. Furthermore, it is also possible to avoid useless
medication and to present indicators for lifestyle habit for health
care, and therefore the present invention is industrially useful.
Sequence CWU 1
1
11655PRTHomo sapiens 1Met Ser Ser Ser Asn Val Glu Val Phe Ile Pro
Val Ser Gln Gly Asn 1 5 10 15 Thr Asn Gly Phe Pro Ala Thr Ala Ser
Asn Asp Leu Lys Ala Phe Thr 20 25 30 Glu Gly Ala Val Leu Ser Phe
His Asn Ile Cys Tyr Arg Val Lys Leu 35 40 45 Lys Ser Gly Phe Leu
Pro Cys Arg Lys Pro Val Glu Lys Glu Ile Leu 50 55 60 Ser Asn Ile
Asn Gly Ile Met Lys Pro Gly Leu Asn Ala Ile Leu Gly 65 70 75 80 Pro
Thr Gly Gly Gly Lys Ser Ser Leu Leu Asp Val Leu Ala Ala Arg 85 90
95 Lys Asp Pro Ser Gly Leu Ser Gly Asp Val Leu Ile Asn Gly Ala Pro
100 105 110 Arg Pro Ala Asn Phe Lys Cys Asn Ser Gly Tyr Val Val Gln
Asp Asp 115 120 125 Val Val Met Gly Thr Leu Thr Val Arg Glu Asn Leu
Gln Phe Ser Ala 130 135 140 Ala Leu Arg Leu Ala Thr Thr Met Thr Asn
His Glu Lys Asn Glu Arg 145 150 155 160 Ile Asn Arg Val Ile Gln Glu
Leu Gly Leu Asp Lys Val Ala Asp Ser 165 170 175 Lys Val Gly Thr Gln
Phe Ile Arg Gly Val Ser Gly Gly Glu Arg Lys 180 185 190 Arg Thr Ser
Ile Gly Met Glu Leu Ile Thr Asp Pro Ser Ile Leu Phe 195 200 205 Leu
Asp Glu Pro Thr Thr Gly Leu Asp Ser Ser Thr Ala Asn Ala Val 210 215
220 Leu Leu Leu Leu Lys Arg Met Ser Lys Gln Gly Arg Thr Ile Ile Phe
225 230 235 240 Ser Ile His Gln Pro Arg Tyr Ser Ile Phe Lys Leu Phe
Asp Ser Leu 245 250 255 Thr Leu Leu Ala Ser Gly Arg Leu Met Phe His
Gly Pro Ala Gln Glu 260 265 270 Ala Leu Gly Tyr Phe Glu Ser Ala Gly
Tyr His Cys Glu Ala Tyr Asn 275 280 285 Asn Pro Ala Asp Phe Phe Leu
Asp Ile Ile Asn Gly Asp Ser Thr Ala 290 295 300 Val Ala Leu Asn Arg
Glu Glu Asp Phe Lys Ala Thr Glu Ile Ile Glu 305 310 315 320 Pro Ser
Lys Gln Asp Lys Pro Leu Ile Glu Lys Leu Ala Glu Ile Tyr 325 330 335
Val Asn Ser Ser Phe Tyr Lys Glu Thr Lys Ala Glu Leu His Gln Leu 340
345 350 Ser Gly Gly Glu Lys Lys Lys Lys Ile Thr Val Phe Lys Glu Ile
Ser 355 360 365 Tyr Thr Thr Ser Phe Cys His Gln Leu Arg Trp Val Ser
Lys Arg Ser 370 375 380 Phe Lys Asn Leu Leu Gly Asn Pro Gln Ala Ser
Ile Ala Gln Ile Ile 385 390 395 400 Val Thr Val Val Leu Gly Leu Val
Ile Gly Ala Ile Tyr Phe Gly Leu 405 410 415 Lys Asn Asp Ser Thr Gly
Ile Gln Asn Arg Ala Gly Val Leu Phe Phe 420 425 430 Leu Thr Thr Asn
Gln Cys Phe Ser Ser Val Ser Ala Val Glu Leu Phe 435 440 445 Val Val
Glu Lys Lys Leu Phe Ile His Glu Tyr Ile Ser Gly Tyr Tyr 450 455 460
Arg Val Ser Ser Tyr Phe Leu Gly Lys Leu Leu Ser Asp Leu Leu Pro 465
470 475 480 Met Arg Met Leu Pro Ser Ile Ile Phe Thr Cys Ile Val Tyr
Phe Met 485 490 495 Leu Gly Leu Lys Pro Lys Ala Asp Ala Phe Phe Val
Met Met Phe Thr 500 505 510 Leu Met Met Val Ala Tyr Ser Ala Ser Ser
Met Ala Leu Ala Ile Ala 515 520 525 Ala Gly Gln Ser Val Val Ser Val
Ala Thr Leu Leu Met Thr Ile Cys 530 535 540 Phe Val Phe Met Met Ile
Phe Ser Gly Leu Leu Val Asn Leu Thr Thr 545 550 555 560 Ile Ala Ser
Trp Leu Ser Trp Leu Gln Tyr Phe Ser Ile Pro Arg Tyr 565 570 575 Gly
Phe Thr Ala Leu Gln His Asn Glu Phe Leu Gly Gln Asn Phe Cys 580 585
590 Pro Gly Leu Asn Ala Thr Gly Asn Asn Pro Cys Asn Tyr Ala Thr Cys
595 600 605 Thr Gly Glu Glu Tyr Leu Val Lys Gln Gly Ile Asp Leu Ser
Pro Trp 610 615 620 Gly Leu Trp Lys Asn His Val Ala Leu Ala Cys Met
Ile Val Ile Phe 625 630 635 640 Leu Thr Ile Ala Tyr Leu Lys Leu Leu
Phe Leu Lys Lys Tyr Ser 645 650 655
* * * * *