U.S. patent application number 14/542633 was filed with the patent office on 2015-03-19 for method for evaluating urate transport-related disease factor and inflammation-related disease factor.
The applicant listed for this patent is HIROTAKA MATSUO, TAKAHIRO NAKAMURA, NARIYOSHI SHINOMIYA, TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCES, THE UNIVERSITY OF TOKYO. Invention is credited to Kimiyoshi Ichida, Yuki Ikebuchi, Kousei Ito, HIROTAKA MATSUO, Takahiro Nakamura, Nariyoshi Shinomiya, Hiroshi Suzuki, Tappei Takada.
Application Number | 20150080257 14/542633 |
Document ID | / |
Family ID | 43386310 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150080257 |
Kind Code |
A1 |
MATSUO; HIROTAKA ; et
al. |
March 19, 2015 |
METHOD FOR EVALUATING URATE TRANSPORT-RELATED DISEASE FACTOR AND
INFLAMMATION-RELATED DISEASE FACTOR
Abstract
A method and evaluation kit are provided, in which a
high-capacity urate transporter is identified to assist in the
early treatment and prevention of urate transport-related disease
and inflammation-related disease. The method can include a step for
detecting variations in genes that encode ABCG2 protein. When a
subject has an SNP of V12M, R113X, Q126X, Q141K, F2085, G268R,
E334X, S441N, L447V, S486N, F506SfsX4, R575X, and/or C608X, it can
be concluded that the subject has a factor that is capable of
inducing urate transport failure, or a state or disease
attributable to that failure. When a subject has an SNP of V12M, it
can be concluded that, unlike the other SNPs, there is a
possibility that the subject does not possess such a factor
because, although this variation itself does not lead to a change
in urate transport capability, said variation is related to linkage
disequilibrium with other SNPs.
Inventors: |
MATSUO; HIROTAKA; (Saitama,
JP) ; Shinomiya; Nariyoshi; (Saitama, JP) ;
Nakamura; Takahiro; (Saitama, JP) ; Takada;
Tappei; (Tokyo, JP) ; Suzuki; Hiroshi; (Tokyo,
JP) ; Ikebuchi; Yuki; (Tokyo, JP) ; Ito;
Kousei; (Tokyo, JP) ; Ichida; Kimiyoshi;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MATSUO; HIROTAKA
SHINOMIYA; NARIYOSHI
NAKAMURA; TAKAHIRO
THE UNIVERSITY OF TOKYO
TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCES |
SAITAMA
SAITAMA
SAITAMA
TOKYO
TOKYO |
|
JP
JP
JP
JP
JP |
|
|
Family ID: |
43386310 |
Appl. No.: |
14/542633 |
Filed: |
November 16, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13379346 |
Jun 19, 2012 |
8940286 |
|
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PCT/JP2010/004154 |
Jun 22, 2010 |
|
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14542633 |
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Current U.S.
Class: |
506/9 ; 506/12;
506/7 |
Current CPC
Class: |
A61P 9/10 20180101; G01N
33/566 20130101; A61P 29/00 20180101; A61K 38/177 20130101; C12Q
2600/106 20130101; G01N 2800/32 20130101; C12Q 2600/156 20130101;
G01N 2800/34 20130101; A61P 19/06 20180101; A61P 13/12 20180101;
A61P 19/02 20180101; C07K 14/705 20130101; A61P 15/00 20180101;
C12Q 1/6883 20130101; C12Q 2600/118 20130101; G01N 33/6893
20130101; G01N 2800/24 20130101 |
Class at
Publication: |
506/9 ; 506/7;
506/12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 22, 2009 |
JP |
2009-148106 |
Claims
1. A method for evaluating urate transport-related disease factor
and inflammation-related disease factor, the method comprising:
evaluating whether or not a subject has a factor that induces urate
transport failure, or a state or a disease attributable to that
failure, the method of evaluating including detecting variations in
genes that encode an ABCG2 protein using a sample containing human
genes of the subject.
2. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 1, wherein the
detecting of variations in genes that encode an ABCG2 protein
includes detecting an SNP or a gene polymorphism having a
relationship of linkage disequilibrium with the SNP.
3. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 2, wherein the
detecting of the gene polymorphism includes using a detection
method selected from the group consisting of 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
SmartAmp method, a Q-probe method (QP method), a MALDI-TOF/MS
method, a molecular beacon method, an RCA method, a UCAN method,
and a nucleic acid hybridization method using a DNA chip or a DNA
microarray.
4. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 1, wherein the
detecting includes identifying an SNP producing at least one amino
acid variation of a component selected from the group consisting of
V12M, R113X, Q126X, Q141K, F208S, G268R, E334X, S441N, L447V,
S486N, F506SfsX4, R575X, and C608X as a factor in the inducement of
urate transport failure, or a state or a disease attributable to
that failure.
5. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 4, wherein the
detecting includes identifying an SNP producing either amino acid
variation of Q126X alone or a combination of Q126X and Q141K as a
factor in the inducement of urate transport failure, or a state or
a disease attributable to that failure.
6. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 1, wherein the
detecting includes identifying a functional change of ABCG2
including a functional failure thereof, as a factor in the
inducement of urate transport failure, or a state or a disease
attributable to that failure.
7. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 6, wherein the
functional change of ABCG2, including a functional failure thereof,
comprises any one of a functional change of ABCG2 by a gene
variation other than the amino acid variations of an SNP producing
at least one amino acid variation of a component selected from the
group consisting of V12M, R113X, Q126X, Q141K, F208S, G268R, E334X,
S441N, L447V, S486N, F506SfsX4, R575X, and C608X, a functional
change of ABCG2 based on a change of an expression amount by a gene
variation in exons or introns containing a promoter and an
untranslated region (UTR) of ABCG2, a functional change of ABCG2 by
a change of a control factor including a transcription factor, or a
compound, a functional change of ABCG2 by a copy number variant
(CNV), an epigenetic change including DNA methylation, a functional
change of ABCG2 by an RNA including a micro RNA or a noncoding RNA,
and a functional change of ABCG2 by a change of a stabilization
mechanism of the ABCG2 protein.
8. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 1, wherein the
detecting includes identifying a serum uric acid level as a state
or a disease attributable to urate transport failure.
9. The method for evaluating urate transport-related disease factor
and inflammation-related disease factor of claim 8, wherein the
serum uric acid level ranges between 6.0 and 9.0 mg/dl.
10. The method for evaluating urate transport-related disease
factor and inflammation-related disease factor of claim 8, wherein
the serum uric acid level ranges between 7.0 and 8.0 mg/dl.
11. The method for evaluating urate transport-related disease
factor and inflammation-related disease factor of claim 1, the
urate transport failure, or state or a disease attributable to that
failure, includes a hyperuricemia selected from a group consisting
of a uric acid overproduction type, an extrarenal uric acid
underexcretion type, a renal uric acid underexcretion type, and a
mixed type thereof.
12. The method for evaluating urate transport-related disease
factor and inflammation-related disease factor of claim 1, wherein
the urate transport failure, or state or a disease attributable to
that failure, includes hyperuricemia, gout, rheumatoid arthritis,
osteoarthritis, infertility, cerebral stroke, an ischemic heart
disease, arrhythmia, photosensitivity, and a chronic kidney
disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/379,346, having a filing date of Jun. 19, 2012, under 35 USC
371 (c) as the National Stage of PCT Application No.
PCT/JP2010/004154, filed Jun. 22, 2010, which claims the benefit of
Japanese Application No. 2009-148106, filed Jun. 22, 2009, each
application of which is incorporated herein by reference in its
entirety.
SEQUENCE LISTINGS
[0002] This application incorporates by reference in its entirety
the last-filed sequence listing submitted in ASCII format via
EFS-Web in U.S. application Ser. No. 13/379,346, filed Jun. 19,
2012. Said ASCII copy, created Jun. 18, 2012, is named TSUBP002.txt
and is 22,058 bytes in size.
TECHNICAL FIELD
[0003] The present invention relates to a urate transporter, as
well as, a method for evaluating urate transport-related disease
factor and inflammation-related disease factor relating to the
transporter, an evaluation kit which implements the method, and
also a test sample and a drug relating to the method and kit.
BACKGROUND ART
[0004] Gout patients have recently increased and the onset age has
become younger. Gout is a disease caused by tissue deposition of
monosodium urate crystals, and often has the onset as a result of
inflammation of the joint. Also, gout is frequently found in
hyperuricemia patients, and it has long been known to have a
heritable component. Gout is often associated with hypertension,
obesity, diabetes, coronary artery diseases, cerebrovascular
diseases, kidney diseases and the like. Also, inflammation-related
diseases include rheumatoid arthritis, infertility and the like,
and early treatment and prevention of these diseases are
needed.
[0005] The present inventors have demonstrated that
loss-of-function mutations in two urate transporter genes, i.e.,
urate transporter 1 (URAT1/SLC22A12) and glucose transporter 9
(GLUT9/SLC2A9), cause renal hypouricemia using function-based
genetic analysis (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.
[0006] However, other urate transporters have not been identified
so far by such analysis, and urate transporters that increase the
serum uric acid (SUA) level and have main pathogenic mutations
causing gout or hyperuricemia remain unidentified.
[0007] The prior art relating to a urate transporter is disclosed
in Patent Literature 1, and the prior arts relating to ABCG2 as a
transporter are disclosed in 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 nor in urate
transport-related disease factor and inflammation-related disease
factor.
CITATION LIST
[0008] Patent Literature 1: JP-A-2003-93067, "Renal and placental
urate transporters and their genes".
[0009] Patent Literature 2: JP-A-2007-60967, "Detection method of
gene polymorphisms and screening method of drugs".
[0010] 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".
[0011] Patent Literature 4: JP-A-2005-529618, "Prediction method of
drug transport capability by ABCG2 polymorphism".
[0012] 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.
[0013] 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.
[0014] 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.
[0015] Non-Patent Literature 4: Maekawa K, Itoda M, Sai K, et al.,
"Genetic variation and haplotype structure of the ABC transporter
gene ABCG2 in a Japanese population", Drug Metab Pharmacokinet
2006; 21:109-21.
[0016] Non-Patent Literature 5: Wang H, Lee E W, Cai X, Ni Z, Zhou
L, Mao Q., "Membrane topology of the human breast cancer resistance
protein (BCRP/ABCG2) determined by epitope insertion and
immunofluorescence", Biochemistry 2008; 47:13778-87.
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0017] Accordingly, the object of the present invention is to
provide a method for evaluating urate transport-related disease
factor and inflammation-related disease factor and to provide an
evaluation kit which implements the method, and also a test sample
and a drug relating to the method and kit so that a high-capacity
urate transporter is identified in order to contribute to the early
treatment and prevention of urate transport-related diseases and
inflammation-related diseases on the basis of the identified
transporter.
Solution to Problem
[0018] The urate transporter according to the present invention is
characterized in that it is formed from proteins having ABCG2 and
is capable of ATP-dependently exporting urate. Preferably, the
transporter does not have at least a single nucleotide polymorphism
(SNP) of Q126X.
[0019] The method for evaluating urate transport-related disease
factor and inflammation-related disease factor according to the
present invention is a method for evaluating whether or not the
subject has a factor that is capable of inducing urate transport
failure, or a state or disease attributable to that failure, the
method including a step of detecting variations in genes that
encode an ABCG2 protein using a sample containing human genes of
the subject. The urate transport-related disease factor and
inflammation-related disease factor strictly mean urate
transport-related disease factor and/or inflammation-related
disease factor.
[0020] The detection of variations in genes that encode an ABCG2
protein may be detection of an SNP or a gene polymorphism having a
relationship of linkage disequilibrium with the SNP.
[0021] For the detection of a gene polymorphism, any one of 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 SmartAmp method, a Q-probe method (QP method), a
MALDI-TOF/MS method, a molecular beacon method, an RCA method, a
UCAN method, and a nucleic acid hybridization method using a DNA
chip or a DNA microarray is useful.
[0022] Subjects may be, for example, a Japanese population, a
population of African descent, and a Caucasian population. The
present invention can be applied similarly to the Pacific Rim
population and other races. When the subject has at least one SNP
of V12M, R113X, Q126X, Q141K, F208S, G268R, E334X, S441N, L447V,
S486N, F506SfsX4, R575X, and/or C608X, it can be concluded that the
subject has a factor that is capable of inducing urate transport
failure, or a state or disease attributable to that failure. When
the subject has an SNP of V12M, it can be concluded indirectly
that, unlike the other SNPs, there is a possibility that the
subject does not possess a factor that is capable of inducing urate
transport failure, or a state or disease attributable to that
failure because, although this variation itself does not lead to a
change in urate transport capability, the variation is related to
linkage disequilibrium with other SNPs. In particular, when the
subject has an SNP of Q126X alone or a combination of Q126X and
Q141K, it can be concluded that the subject has a factor that is
capable of inducing urate transport failure, or a state or disease
attributable to that failure. Also, when the subject has a
functional change of ABCG2 including a functional failure thereof
without being limited to SNPs producing the above amino acid
variations, it can be concluded that the subject has a factor that
is capable of inducing urate transport failure, or a state or
disease attributable to that failure.
[0023] Examples of such a functional change of ABCG2 including a
functional failure thereof include a functional change of ABCG2 by
a gene variation other than the above amino acid variations, a
functional change of ABCG2 based on a change of an expression
amount and the like by a gene variation in exons and introns
containing a promoter and an untranslated region (UTR) of ABCG2, a
functional change of ABCG2 by a change of a regulating factor such
as a transcription factor, a compound and the like, a functional
change of ABCG2 by CNV (copy number variant), an epigenetic change
including DNA methylation, a functional change of ABCG2 by an RNA
including a micro RNA and a noncoding RNA, and a functional change
of ABCG2 by a change of a stabilization mechanism of the ABCG2
protein.
[0024] When a serum uric acid level is a given level or more, it
can be concluded that the subject highly has a factor that is
capable of inducing urate transport failure, or a state or disease
attributable to that failure. The threshold level of the serum uric
acid level is preferably any level between 6.0 and 9.0 mg/dl such
as, for example, 6.6, 7.0 and 8.0 mg/dl, and more preferably
between 7.0 and 8.0 mg/dl.
[0025] Also, hyperuricemia may be classified into a uric acid
overproduction type, an extrarenal uric acid underexcretion type, a
renal uric acid underexcretion type, and a mixed type thereof, and
classification of hyperuricemia may be identified on the basis of
an evaluation of an ABCG2 function so as to contribute to treatment
depending on its cause. In this case, findings in urine and blood
may be considered concomitantly.
[0026] Examples of the urate transport-related diseases and
inflammation-related diseases include hyperuricemia, gout,
rheumatoid arthritis, osteoarthritis, infertility, cerebral stroke,
ischemic heart disease, arrhythmia, photosensitivity, chronic
kidney disease and the like.
[0027] The evaluation kit for urate transport-related disease
factor and inflammation-related disease factor according to the
present invention is a kit for evaluating whether or not the
subject has a factor that is capable of inducing urate transport
failure, or a state or disease attributable to that failure, the
kit including: means for detecting at least one SNP of V12M, R113X,
Q126X, Q141K, F208S, G268R, E334X, S441N, L447V, S486N, F506SfsX4,
R575X, and C608X in an ABCG2 gene, or a gene polymorphism having a
relationship of linkage disequilibrium with the SNP, using a sample
containing human genes of the subject.
[0028] The nonhuman animals according to the present invention are
those for examining urate transport kinetics, and are characterized
in that they have a deficiency of an ABCG2 gene.
[0029] The method for examining urate transport kinetics according
to the present invention uses nonhuman animals having a deficiency
of an ABCG2 gene, and may measure their serum uric acid levels.
Similarly, the method can be carried out using nonhuman animals
overexpressing a human ABCG2 gene or a nonhuman ABCG2 gene,
nonhuman animals overexpressing a human ABCG2 gene or a nonhuman
ABCG2 gene containing at least one variation of V12M, R113X, Q126X,
Q141K, F208S, G268R, E334X, S441N, L447V, S486N, F506SfsX4, R575X,
and C608X, nonhuman cell lines or human cell lines having a
deficiency of an ABCG2 gene, nonhuman cell lines or human cell
lines overexpressing a human ABCG2 gene or a nonhuman ABCG2 gene,
nonhuman cell lines or human cell lines overexpressing a human
ABCG2 gene or a nonhuman ABCG2 gene containing at least one
variation of V12M, R113X, Q126X, Q141K, F208S, G268R, E334X, S441N,
L447V, S486N, F506SfsX4, R575X, and C608X, or cell membrane
vesicles prepared from these cell lines. Mice bred using a
feedstuff containing oxonate which is an inhibitor of uricase which
is a urate-metabolizing enzyme are useful as the nonhuman animals
for examining urate transport kinetics.
[0030] The drug for urate transport-related diseases and
inflammation-related diseases according to the present invention is
a drug for reducing a factor that is capable of inducing urate
transport failure, or a state or disease attributable to that
failure, the drug containing: a polynucleotide encoding an ABCG2
protein in the form capable of introducing it into cells.
[0031] Similarly, the drug according to the present invention is a
drug for reducing a factor that is capable of inducing urate
transport failure, or a state or disease attributable to that
failure, the drug may include: a polypeptide corresponding to an
ABCG2 protein in the form capable of introducing it into cells.
Effects of the Invention
[0032] The present invention provides a high-capacity urate
transporter, and concomitantly contributes to early treatment and
prevention of urate transport-related diseases.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIGS. 1A and 1B provide an explanatory diagram of primers
(SEQ ID NOs:1-61) for mutation analysis designed on the basis of
gene structure of human ABCG2 gene.
[0034] FIG. 2 is a graph showing [.sup.3H]ES transport plotted
against inhibitory substances.
[0035] FIG. 3A is a graph showing [.sup.14C]urate transport against
time, and FIG. 3B is a graph showing [.sup.14C]urate transport
plotted against urate concentration.
[0036] FIG. 4 shows a topology model of human ABCG2 (SEQ ID NO:62)
and the nonsynonymous mutation sites found in hyperuricemia
patients.
[0037] FIGS. 5A-5E show the results of sequence analysis of ABCG2
(SEQ ID NOs:63-90).
[0038] FIG. 6 is a graph showing the results of urate transport
analysis of mutated ABCG2.
[0039] FIGS. 7A-7C are graphs showing the results of quantitative
trait locus (QTL) analysis of Q141K, and FIG. 7A is for male and
female, FIG. 7B for male, and FIG. 7C for female.
[0040] FIG. 8 shows a urate excretion model in kidney, liver and
intestine.
[0041] FIG. 9 is a table showing the appearance frequency of an
estimated functional decline of ABCG2 in general residents (health
check examinees).
[0042] FIG. 10 is a table showing the association of a functional
decline of ABCG2 in male gout patients.
[0043] FIG. 11 is a graph showing a relationship between the ABCG2
function and the onset age.
[0044] FIG. 12 is a table showing the racial differences in respect
of various ABCG2 variants.
[0045] FIG. 13 is a graph showing the transport of [.sup.14C]urate
via mouse ABCG2.
[0046] FIGS. 14A-14C are graphs showing blood uric acid levels and
urinary uric acid levels in wild-type mice and ABCG2-deficient
mice.
[0047] FIG. 15 is a graph showing a relationship between the ABCG2
function and the urinary uric acid excretion amount in gout and
hyperuricemia patients.
[0048] FIG. 16 is a graph showing the percentage of a traditional
type of clinical classifications in hyperuricemia cases having each
estimated ABCG2 function.
DESCRIPTION OF EMBODIMENTS
[0049] The present inventors have found a high-capacity transporter
of urate as an extension of the findings disclosed in Non-Patent
Literatures 1 and 2 and the like, and thus leading to the present
invention.
[0050] 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 following Examples, and design can be changed by appropriately
using conventionally known techniques.
[0051] Although Japanese individuals are mainly exemplified herein
as the subject, the present invention can be applied similarly 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 focused 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.
[0052] The ATP-binding cassette, subfamily G, member 2 gene
ABCG2/BCRP locates in a gout-susceptibility locus (MIM138900) on
chromosome 4q, and it encodes 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).
[0053] Accordingly, as described below, the present inventors
showed that ABCG2 is the first urate excretion transporter found in
human and that its common variants increase serum uric acid (SUA)
levels, and they performed clinicogenetic analysis of the ABCG2
gene.
[0054] In order to confirm whether or not ABCG2 exerts an adverse
influence on uric acid handling and the onset of gout, a
molecular-function-based clinicogenetic (FBCG) analysis was
performed. High-molecular-weight genomic DNAs were extracted from
all peripheral blood cells taken from subjects. For quantitative
trait locus (QTL) analysis of serum uric acid levels, genotyping of
the dysfunctional common variant Q141K in 739 Japanese individuals
was performed. To examine a frequency of a functional decline of
ABCG2, genotyping of ABCG2 was performed in another 2150 Japanese
health check examinees (1042 male individuals, 1108 female
individuals).
[0055] For association studies, 228 Japanese male hyperuricemia
cases (including 161 gout cases) as well as more than several
hundreds of Japanese male controls (SUA 7.0 mg/dl) were genotyped.
For gout, more than 700 male cases and more than 1800 Japanese male
controls (SUA 7.0 mg/dl) were genotyped.
[0056] Female gout cases and hyperuricemia cases were also
analyzed. All gout patients were clinically diagnosed as primary
gout. Individuals whose serum uric acid levels had been more than
8.0 mg/dl were selected as hyperuricemia cases. To examine the
presence and frequency of a functional decline of ABCG2 in
individuals other than Japanese individuals, genotyping was also
performed in 199 Caucasian individuals and 98 individuals of
African descent.
[0057] Wild-type ABCG2 cDNA was inserted into the Nhe I site and
Apa I site of pcDNA3.1(+) vector plasmid (Invitrogen, Carlsbad,
Calif.), with a myc-tag sequence attached at the 5' end. To prepare
membrane vesicles, HEK293 cells were transiently transfected with
an expression vector for ABCG2 or an empty vector using FuGENE6
(Roche Diagnostics, Indianapolis, Ind.). Forty-eight hours later,
cells were harvested and the membrane vesicles were isolated using
a standard method. The uptake study of [.sup.3H]estrone-3-sulfate
(ES, 500 nM) and [.sup.14C]urate (28 .mu.M) was performed.
[0058] Using the site-directed mutagenesis technique, mutants of
ABCG2 (V12M, R113X, Q126X, Q141K, F208S, G268R, E334X, S441N,
L447V, S486N, F506SfsX4, R575X, C608X) were constructed on the
expression vector for ABCG2, and used for urate transport analysis.
Western blot analysis of the membrane vesicles (20 .mu.g) was
performed using an 800-fold diluted anti-myc-tag antibody (Roche
Diagnostics).
[0059] In order to find candidate variants in ABCG2, mutation
analysis of all coding regions and intron-exon boundaries of the
ABCG2 gene was performed for 80 Japanese hyperuricemia
patients.
[0060] FIGS. 1A and 1B provide an explanatory diagram of primers
(SEQ ID NOs:1-61) for mutation analysis designed on the basis of
gene structure of human ABCG2 gene. Genomic DNA was amplified by
PCR with these primers. Base sequences of the PCR products were
analyzed using a 3130.times.1 Genetic Analyzer (Applied Biosystems,
Carlsbad, Calif.). Genotyping was also performed by an allelic
discrimination assay (Custom Taqman MGB, Applied Biosystems) with a
7700 detector (Applied Biosystems) or melting analysis (HRM method)
with LightCycler 480 (Roche Diagnostics).
[0061] For all calculations of statistical analysis, the software R
and SPSS (SPSS Japan Inc.) were used. The differences in the
clinical covariates between the various genotypes of the SNPs of
ABCG2 were compared using Mann-Whitney and Kruskal-Wallis tests.
The Chi-square test and Fisher's exact test were used to compare
the difference in genotype frequencies and allele frequencies
between the gout cases and control samples. Haplotype estimation
was performed using the EM algorithm. Examination of a risk of
diseases such as gout due to a functional decline of ABCG2 was
evaluated using logistic regression analysis.
[0062] Using membrane vesicles prepared from ABCG2-expressing
cells, the inhibitory effect of urate on ABCG2-mediated transport
of its typical substrate, ES (estrone-3-sulfate) was examined.
[0063] FIG. 2 is a graph showing [.sup.3H]ES transport plotted
against inhibitory substances. The inhibitory effect on the
transport of [.sup.3H]estrone-3-sulfate (ES, 500 nM), a typical
substrate of ABCG2 was examined using the vesicle transport assay
system. In addition to ES, the inhibition by another substrate,
3'-azido-3'-deoxythymidine (AZT) was observed. ES transport was
also inhibited by urate, which suggests the possibility of urate
transport via ABCG2.
[0064] In order to demonstrate whether or not urate is a substrate
of ABCG2, transport assays were performed using isotope-labeled
[.sup.14C]urate.
[0065] FIG. 3A is a graph showing [.sup.14C]urate transport against
time, and FIG. 3B is a graph showing [.sup.14C]urate transport
plotted against urate concentration. As shown in FIG. 3A, an
ATP-dependent urate transport was detected in ABCG2-expressing
vesicles but not in control vesicles. This is the first evidence of
a direct high-capacity urate transport via ABCG2. Because of a mild
inhibitory effect on the ES transport, urate was assumed to be a
high-capacity substrate of ABCG2. Indeed, as shown in FIG. 3B,
ABCG2-mediated urate transport scarcely reached saturation at
concentrations of 1 mM or less.
[0066] Typical ABCG2 substrates, e.g., sulfate conjugates such as
ES, 4-methylumbelliferone sulfate, and E3040 sulfate, are
transported by ABCG2 with low capacity (K.sub.m value of about 20
.mu.M). Kinetic analysis revealed that ABCG2 mediated the saturable
transport of urate with a K.sub.m of 8.24.+-.1.44 mM and a
V.sub.max of 6.96.+-.0.89 nmol/min/mg (protein), and therefore, it
can be said that an ABCG2-mediated high-capacity transport remains
functional under a high-urate condition.
[0067] These findings reasonably explain a newly identified
physiological role of ABCG2 as a high-capacity urate exporter.
[0068] FIG. 4 shows a topology model of human ABCG2 and the
nonsynonymous mutation sites found in hyperuricemia patients, and
FIGS. 5A-5E show the results of sequence analysis of ABCG2 (SEQ ID
NOs:63-90).
[0069] Base sequences of all coding regions of the ABCG2 gene were
analyzed in 80 hyperuricemia patients, and five mutations with
amino acid alterations (V12M, Q126X, Q141K, S441N, F506SfsX4) were
found. "#" represents an N-linked glycosylation site (N596), and
"*" represents cysteine residues for disulfide bonds (C592, C603
and C608).
[0070] V12M, Q126X and Q141K are SNPs present in the intracellular
N-terminal region. It is reported that allele frequencies for these
SNPs, which are quite common in the Japanese population, were 31.9%
for Q141K, 19.2% for V12M, and 2.8% for Q126X (Non-Patent
Literature 4). Calculations of these data on the basis of
Hardy-Weinberg's equilibrium revealed that estimates of the
frequencies of Japanese individuals with these minor alleles were
53.6% for Q141K, 34.7% for V12M, and 5.5% for Q126X. The topology
model as shown in the figure is based on the recent report for
membrane topology determination of human ABCG2 (Non-Patent
Literature 5).
[0071] FIG. 6 is a graph showing the results of urate transport
analysis of mutated ABCG2. In order to clarify the effect of urate
transport activities on ABCG2 function, the activities of mutants
were examined using membrane vesicles expressing wild-type and
mutant ABCG2 proteins.
[0072] ATP-dependent urate transport was reduced by approximately
half (46.7%) in Q141K and was nearly eliminated in Q126X, G268R,
S441N, and F506SfsX4 mutants. Western blot analysis showed that
ABCG2 protein expression in the Q141K variant decreased by half
(45.2%), while Q126X showed no protein expression on membrane
vesicles. Also, ATP-dependent urate transport of ABCG2 was
remarkably reduced by F208S, E334X, L447V, S486N, R575X, and C608X
mutations, and was nearly eliminated in F208S, E334X, L447V, S486N,
and R575X mutants.
[0073] The half-decreased urate transport activity of Q141K may be
ascribed to the half-decreased expression of ABCG2 protein, which
is consistent with the disclosure of Non-Patent Literature 3 on ES
transport.
[0074] While loss of urate transport in the Q126X mutant should be
caused by the complete lack of protein expression, V12M did not
show any changes in urate transport and in protein expression
relative to wild-type ABCG2. These data clearly show that the
degree of decreased ABCG2 protein expression directly affects the
urate transport activity.
[0075] FIGS. 7A-7C are graphs showing the results of quantitative
trait locus (QTL) analysis of Q141K, and FIG. 7A is for male and
female, FIG. 7B for male, and FIG. 7C for female. Quantitative
trait locus (QTL) analysis of serum uric acid levels was performed
with the high-frequency dysfunctional variant Q141K in ABCG2, for
739 Japanese individuals including 245 male subjects and 494 female
subjects. "C/C", "C/A", and "NA" indicate wild-type subjects,
heterozygous mutation carriers, and homozygous mutation carriers of
Q141K, respectively.
[0076] Serum uric acid levels significantly increased as the minor
alleles of Q141K increased (p=6.00.times.10.sup.-5, FIG. 7 (A)). A
significant increase in the serum uric acid levels was observed in
both male (p=0.0144) and female (p=0.0137) subjects. Also, Q141K
had no significant association with other clinical parameters such
as age, body mass index, or sex.
[0077] These findings indicate that ABCG2 has a physiological
function to decrease the serum uric acid levels, and that there
could be great inter-individual differences in its function
resulting from SNPs of ABCG2.
[0078] FIG. 8 is an explanatory diagram showing a urate excretion
model in kidney, liver and intestine. Two-thirds of uric acid in
the body is normally excreted through the kidney, while one-third
gains entrance to the gut where it undergoes uricolysis. In the
human kidney, urate is bi-directionally reabsorbed and secreted via
urate transporters.
[0079] ABCG2 is expressed on the apical side of proximal tubular
cells (kidney) and of hepatocytes (liver), and enterocytes
(intestine). In an impaired model, common SNPs in ABCG2 on the
apical side reduce the urate excretion and elevate the serum uric
acid levels. Based on this impaired model, a physiological urate
excretion model is proposed in which ABCG2 mediates renal urate
excretion via urinary secretion.
[0080] In this model, it is also considered that ABCG2 mediates gut
urate excretion via biliary and intestinal secretion. In proximal
tubular cells, other urate transporters (URAT1 and GLUT9) mediate
renal urate reabsorption. The location of GLUT9L (GLUT9 isoform 1)
and GLUT9S (GLUT9 isoform 2) is based on observations from
polarized MDCK (Madin-Darby canine kidney) cells.
[0081] Genotyping of ABCG2 SNPs for 228 Japanese male hyperuricemia
cases (including 161 gout cases) was performed. If minor alleles
are allele 1 and major alleles are allele 2, allele 1 is T and
allele 2 is C in Q126X, allele 1 is A and allele 2 is C in Q141K,
and allele 1 is A and allele 2 is G in V12M. It was found that
Q126X significantly increased gout risk. Also, the dysfunctional
SNP, Q141K significantly increased gout risk. Either of these
mutations was observed in 80% or more gout cases. A similar
observation was also recognized in an association analysis of
hyperuricemia cases. Also, gout patients with Q126X homozygous
mutations were observed, and furthermore, cases with Q126X
homozygous mutations were also observed in asymptomatic
hyperuricemia without gout. The serum uric acid level was 10 mg/dl
or more in both cases.
[0082] In addition, haplotype frequency analysis of V12M, Q126X,
and Q141K revealed that there is no simultaneous presence of the
minor genes Q126X and Q141K in one haplotype. The haplotype with
Q126X markedly increases gout risk as compared with non-risk
haplotypes. Q141K is assigned to another independent risk
haplotype. Thus, Q126X and Q141K are independent risk factors, and,
merely by examining an SNP of each Q126X or Q141K, it is possible
to evaluate easily whether or not a haplotype with its presence is
a risk haplotype.
[0083] Also, it was found that, when the subject has a minor gene
V12M (an SNP of V12M), it can be concluded indirectly that, unlike
the other SNPs, there is a possibility that the subject does not
possess a factor that is capable of inducing urate transport
failure, or a state or disease attributable to that failure
because, although this variation itself does not lead to a change
in urate transport capability, the variation is related to linkage
disequilibrium with other SNPs.
[0084] FIG. 9 is a table showing the appearance frequency of an
estimated functional decline of ABCG2 in general residents (health
check examinees). The functional decline of ABCG2 was recognized in
more than half of examinees, but one-fourth or less functional
decline was recognized only by about 1.2 to 1.7%. The estimated
functional decline of ABCG2 was recognized similarly in male and
female individuals.
[0085] FIG. 10 is a table showing the association of a functional
decline of ABCG2 in male gout patients. It is clear that the onset
risk becomes higher as the ABCG2 function declines. As shown in
FIG. 10, the functional decline of ABCG2 was recognized in about
80% of gout cases, and 2.7-fold or more elevation of gout risk was
recognized. In about 30% of gout cases, one-half or less decline of
ABCG2 function was recognized, and 4.8-fold or more elevation of
gout risk was recognized. Furthermore, one-fourth or less decline
of function was recognized in 5% or more gout cases, and 10-fold or
more increase of risk was recognized. It was found that significant
increase of gout onset risk is recognized even in mild functional
decline, and that the onset risk markedly increases as the
functional decline is greater. Also in analysis of female gout
cases, the functional decline of ABCG2 was recognized in many
cases, which suggested that the decline is involved in the onset of
gout.
[0086] FIG. 11 is a graph showing a relationship between the ABCG2
function and the onset age. Analysis of more than 700 gout cases
revealed that the onset age of gout becomes younger as the ABCG2
function declines. It was found that, when the ABCG2 function is
1/4 below, the onset risk at the young age of twenties and younger
becomes 20-fold greater than a normal risk. It was also found that,
even when the ABCG2 function is 1/2 and 3/4, the gout onset risk at
the young age of twenties and younger is very high.
[0087] The functional decline of ABCG2 is closely related to the
onset of gout at the young age, and therefore, early recognition of
the gout risk is helpful for early prevention of the onset of gout,
as well as for early treatment and prevention of worsening of
symptoms when the gout is developed. Accordingly, analysis of ABCG2
function-declining SNPs and prediction of ABCG2 function based on
the analysis are important to predict onset risk of diseases such
as gout.
[0088] FIG. 12 is a table showing the racial differences in respect
of various ABCG2 variants. Risky variation Q126X is recognized in
many individuals of African descent, and also recognized in
Caucasians. Conversely, Q141K is recognized in less individuals of
African descent, and in more Caucasians. Also, homo variation is
recognized in Caucasian individuals, but not in individuals of
African descent. Accordingly, it was found that analysis of
combination of two variations is very important in individuals of
African descent, and also worthy in Caucasians. In this connection,
there is a possibility that analysis focused on gout cases
increases the frequency.
[0089] Also, it was found that estimated ABCG2 function of 1/4 or
below was recognized in many individuals of African descent rich in
Q126X variation to the same degree as in Japanese individuals. In
individuals of African descent, the function of 3/4 was less likely
to be recognized (about 10%) because they are poor in Q141K as
compared with other races. In Caucasians, the function of 1/4 or
below was less likely to be recognized because they are poor in
Q126X variation, but the function of 3/4 was recognized more
frequently (about 15%) as compared with individuals of African
descent because they are rich in Q141K.
[0090] As is apparent from these results, analysis of ABCG2
function-declining SNPs and prediction of ABCG2 function based on
the analysis are important to predict onset risk of diseases such
as gout, not only in Japanese individuals but also in individuals
of African descent and Caucasians.
[0091] In order to clarify the role of ABCG2 in urate kinetics,
analysis was performed using an animal model. The present inventors
examined using mice whether or not mouse ABCG2 has a urate
transport capability in the same manner as in human ABCG2, by a
transport experiment using cell membrane vesicles.
[0092] Since most mammals other than some primates including human
have urate-metabolizing enzyme, uricase, use of untreated mice is
improper for a model reflecting human urate kinetics. Accordingly,
mice to which a uricase inhibitor, potassium oxonate was daily
administered were used. The administration was performed by
breeding mice using an oxonate-containing feedstuff which was
prepared by adding 2.0% (w/w) potassium oxonate (TokyoChemical
Industry, Tokyo, Japan) to MF feed stuff (Oriental Yeast Co., Ltd.,
Tokyo, Japan).
[0093] A mouse ABCG2 expression vector was constructed by
amplifying a cDNA of mouse ABCG2 with a myc tag sequence attached
to the N-terminus, integrating it into pGEM T-Easy Vector (Promega,
Madison, Wis.), and then integrating it into a Not I site of a
pcDNA3.1(+) vector via a restriction enzyme treatment.
[0094] In order to confirm the expression of mouse ABCG2 via the
myc-mABCG2/pcDNA3.1(+) vector thus prepared, the vector was
transiently introduced into polarized cells, LLC-PK1 cells, and the
localization pattern was observed. The cells were immunostained
using an anti-myc antibody and observed by a confocal microscope.
The results showed that the mouse ABCG2 is localized on the apical
membrane surface of the LLC-PK1 cells, and the results were
consistent with the localization in a living body.
[0095] Also, in order to confirm whether or not the mouse ABCG2
transports urate in the same manner as in human ABCG2, HEK293 cells
into which the mouse myc-ABCG2 expression vector was transiently
introduced were recovered, and cell membrane vesicles were
prepared. In order to confirm the expression of mouse myc-ABCG2,
western blotting was performed, and a band was observed at the
location of about 85 kDa.
[0096] Small gut excised from wild-type FVB mice and
ABCG2-deficient mice (body weight 27-32 g) bred using an
oxonate-containing feedstuff was divided into 3 portions, and a
transport experiment was performed using the most upstream portion.
One end of the gut tract was connected to a 5 ml syringe and the
other end to a 2.5 ml syringe. As a mucosal side solution, 5 ml of
Ringer Buffer previously warmed to 37.degree. C. was introduced
through the 5 ml syringe to fill lumen of the gut tract. Ringer
Buffer at pH 7.4 containing 0.02 .mu.Ci/mI radioisotope-labeled
uric acid (final concentration of radioisotope uric acid 400 nM)
was warmed at 37.degree. C. for 30 minutes with aeration of an
oxygen-carbon dioxide mixed gas, and the experiment was then
started by setting the time point when the gut tract was set to 0
minute.
[0097] FIG. 13 is a graph showing the transport of [14C]urate via
mouse ABCG2. Transport experiments were performed using cell
membrane vesicles and using radioisotope-labeled uric acid as a
substrate. As a result, it was confirmed that the mouse ABCG2 also
transports uric acid in the same manner as in human ABCG2. Also,
transport experiments were performed in uric acid concentrations of
250 .mu.M, 500 .mu.M, 1 mM, 1.5 mM, 2 mM, and 4 mM, respectively.
As a result, no saturability was found in this concentration range.
Whereby, it was shown that mouse ABCG2 is a high-capacity urate
transporter which can function even in the presence of a high
concentration of uric acid.
[0098] FIGS. 14A-14C are graphs showing blood uric acid levels and
urinary uric acid levels in wild-type mice and ABCG2-deficient
mice. Blood uric acid levels were compared between wild-type mice
and ABCG2-deficient mice receiving an oxonate-containing feedstuff
for 2 or more weeks. As a result, it was found that blood uric acid
levels in ABCG2-deficient mice significantly increased as compared
with those in wild-type mice (FIG. 14 (A)). Since the elevation of
blood uric acid levels due to the decline of ABCG2 function was
confirmed in mice in the same manner as in human, the mouse model
can be used as a model reflecting urate kinetics in human. Also,
with a significant elevation of blood uric acid levels (FIG. 14
(B)), urinary uric acid levels also showed an elevation tendency
although it was not significant (FIG. 14 (C)). The ratio of urinary
uric acid levels/blood uric acid levels, corrected using urine
concentrations and blood concentrations of creatinine which serves
as an indicator of a renal function, significantly increased in
ABCG2-deficient mice. The results show that the cause of an
elevation of blood uric acid levels due to an ABCG2 deficiency can
not be explained by a decrease of urinary uric acid excretion
amount.
[0099] Urate transport experiments were performed using the small
intestine isolated from wild-type mice and ABCG2-deficient mice.
Since uric acid secretion from the gut tract is known as a uric
acid excretion pathway other than urinary excretion, the small
intestine was isolated from wild-type mice and ABCG2-deficient
mice, and transport experiments of radioisotope-labeled uric acid
were performed. As a result, a linear urate transport was
recognized up to 30 minutes both in wild-type mice and in
ABCG2-deficient mice, and the transport amount of uric acid at 30
minutes significantly decreased in ABCG2-deficient mice. Whereby,
it was suggested that mouse ABCG2 is involved in the urate
transport in the small intestine.
[0100] FIG. 15 is a graph showing a relationship between the ABCG2
function and the urinary uric acid excretion amount (UUAV) in gout
and hyperuricemia patients (cases diagnosed by physicians). It is
understood that the urinary uric acid excretion amount tends to
increase as the ABCG2 function declines. The increase of the
urinary uric acid excretion amount is a characteristic feature of
hyperuricemia referred to as a uric acid overproduction type.
[0101] FIG. 16 is a graph showing the percentage of a traditional
type of clinical classifications in hyperuricemia cases having each
estimated ABCG2 function. It can be said that the percentage
containing a uric acid overproduction type and a mixed type is high
as the ABCG2 function declines. Also, it can be recognized that
patients having decline of the ABCG2 function are frequently
recognized in the uric acid overproduction type and mixed type (80%
or more), and conversely, patients having decline of the ABCG2
function are poorly recognized in a urinary uric acid
underexcretion type.
[0102] It was found that, in the traditional overproduction type,
any functional decline of ABCG2 is recognized in about 80 to 90% of
the cases. It was also found that, even in the mixed type, any
functional decline of ABCG2 is recognized in about 70 to 80% of the
cases.
[0103] Evaluation of the ABCG2 function enabled a new, more precise
clinical classification of hyperuricemia.
[0104] Thus, it was found that, in fact, many cases handled as the
uric acid overproduction type in a traditional classification are
not caused by the overproduction, but their pathogenesis lies in an
extrarenal uric acid underexcretion caused by a functional decline
of ABCG2. It was found that the cases are a uric acid overexcretion
type in the kidney (renal overexcretion type) just like the
traditional uric acid overproduction type.
[0105] It was found that, in the traditional overproduction type,
any functional decline of ABCG2 is frequently recognized, and
therefore, a type caused by decrease of extrarenal excretion of
uric acid (extrarenal uric acid underexcretion type) constitutes a
majority.
[0106] Previously, it was considered that excretion into urine is
important as a uric acid excretion pathway, and elevation of a
blood uric acid level is mainly caused by decrease of a uric acid
amount excreted into urine and a uric acid overproduction. Also in
clinical practice, the elevation of a blood uric acid level was
considered by classifying into the urinary uric acid underexcretion
type and uric acid overproduction type. Mainstream prediction and
discussion were that ABCG2 assumes a function of a uric acid
excretion in the kidney, and a urinary uric acid excretion
clearance decreases by a deficiency of ABCG2.
[0107] To the contrary, the present inventors showed that, in
ABCG2-deficient mice receiving an oxonate-containing feedstuff, the
ratio of urinary uric acid levels/blood uric acid levels
significantly increased, when corrected using urine concentrations
and blood concentrations of creatinine which serves as an indicator
of a renal function. The results show that an elevation of blood
uric acid levels due to a functional decline of ABCG2 can not be
explained by a uric acid excretion from the kidney, and that the
blood uric acid levels increase due to a decrease of a uric acid
excretion via ABCG2 from organs other than the kidney. Also, they
found that, in patients having blood uric acid levels increased due
to a functional decline of ABCG2, a urinary uric acid excretion
clearance does not decrease but rather shows an increasing
tendency.
[0108] Regarding the excretion pathway other than urine, there is a
report showing that sweat glands excrete only a negligible degree
of uric acid, and it is considered that uric acid is excreted
mainly into feces other than in the pathway for the urinary
excretion. It is considered that, with respect to the pathway
excreted into feces, uric acid secreted from saliva, gastric juice,
and bile is each about 5% or below of uric acid excreted per day
from the body. Accordingly, it is difficult to explain the
elevation of blood uric acid levels even if these pathways are
blocked. From these facts, it is likely that the decrease of uric
acid excretion in the small intestine contributes to the elevation
of blood uric acid levels due to an ABCG2 deficiency. In fact, the
results of transport experiments using the small gut suggested that
ABCG2 is involved in uric acid excretion from the small gut.
[0109] Use of an upstream portion of the small intestine in the
transport experiments using the gut tract is based on a report
showing that the expression of ABCG2 in human is high in an upper
portion of the small intestine. Actually, the results of
experiments performed using a lower portion of the small intestine
also showed a weak urate transport as compared with that of an
upstream portion, and a tendency showing no difference between
wild-type mice and ABCG2-deficient mice was recognized. This
suggests that gut tract secretion of uric acid via ABCG2
corresponds to its expression distribution, and is conducted mainly
in an upper portion of the small gut.
[0110] Involvement of ABCG2 in uric acid excretion from the small
gut suggests that blood uric acid levels can be decreased by
inducing or activating ABCG2 of the digestive tract. Thus, the
suggestion contributes to the development of a new blood uric acid
level-lowering drug capable of using in patients having a renal
failure.
[0111] Also, some hyperuricemia patients classified as those having
a uric acid overproduction type in the traditional classification
have a possibility that the cause is a uric acid underexcretion
from the digestive tract, and therefore, the present invention
contributes to diagnosis and prehension of a precise disease type
of hyperuricemia, to suitable, effective use of therapeutic drugs,
and to the development of therapeutic drugs based on a disease
state.
[0112] Currently, for gout treatment, symptomatic therapy using
NSAIDs is conducted during an attack. In addition, allopurinol
which suppresses uric acid production, benzbromarone, probenecid
and the like which are inhibitory drugs of uric acid reabsorption
is prophylactically used for the purpose of retaining blood uric
acid levels at a lower level. However, drugs accelerating urinary
excretion are accompanied with a risk of urinary calculus as a side
effect. Inhibition of ABCG2 is not desirable for improvement of
hyperuricemia and lowering of a onset risk of gout. Instead, drugs
causing induction of ABCG2 expression and enhancement of ABCG2
function are more suitable. Alternatively, drugs which do not lower
the ABCG2 function but cause lowering of expression of URAT1 and
GLUT9 and inhibition of their functions are more suitable.
[0113] Also, a clinical classification of hyperuricemia and
selection of therapeutic drugs can be practiced more suitably by
typing of SNPs of ABCG2 or evaluation of a uric acid excretion
(detailed evaluation of a uric acid excretion amount in excreta and
simple evaluation of a uric acid excretion pattern in a spot urine,
in the latter case, reliability can be more increased by correcting
on the basis of physical constitutions such as body weight).
[0114] From the above facts, it is identified that a combination of
Q126X variation and other function-declining variation in an ABCG2
gene is a main cause of primary gout. These findings suggest the
importance of non-functional variants of ABCG2 such as Q126X, which
substantially inhibit urate excretion and cause gout.
[0115] Accordingly, the present invention provides, as a
high-capacity urate transporter, a transporter which is formed from
a protein having ABCG2 and is capable of selectively and
ATP-dependently excreting urate, and preferably a transporter
having no function-declining SNP such as at least Q126X.
[0116] Also, a combination of a function-losing variation such as
Q126X and a half function-losing variation (Q141K) plays an
important role in elevation of serum uric acid levels and the onset
of gout. Accordingly, when the subject has one function-losing
variation such as Q126X, in a simple examination, it is also
possible to evaluate that the subject has a factor of urate
transport-related diseases and inflammation-related diseases such
as gout.
[0117] The method for evaluating urate transport-related disease
factor and inflammation-related disease factor according to the
present invention is a method for evaluating whether or not the
subject has a factor that is capable of inducing urate transport
failure, or a state or disease attributable to that failure, the
method including a step for detecting variations in genes that
encode an ABCG2 protein using a sample containing human genes of
the subject.
[0118] Genes encoding the ABCG2 protein include V12M, R113X, Q126X,
Q141K, F208S, G268R, E334X, S441N, L447V, S486N, F506SfsX4, R575X,
and C608X, and, when an SNP or a gene polymorphism having a
relationship of linkage disequilibrium with the SNP is detected in
the subject, it is concluded that the subject has the factor.
[0119] Also, when the subject has a functional change of ABCG2
including a functional failure thereof without being limited to
SNPs producing the above amino acid variations, it can be concluded
that the subject has a factor that is capable of inducing urate
transport failure, or a state or disease attributable to that
failure.
[0120] Examples of such a functional change of ABCG2 including a
functional failure thereof include a functional change of ABCG2 by
a gene variation other than the above amino acid variations, a
functional change of ABCG2 based on a change of an expression
amount and the like by a gene variation in exons and introns
containing a promoter and an untranslated region (UTR) of ABCG2, a
functional change of ABCG2 by a change of a regulating factor such
as a transcription factor, a compound and the like, a functional
change of ABCG2 by CNV (copy number variant), an epigenetic change
including DNA methylation, a functional change of ABCG2 by an RNA
including a micro RNA and a noncoding RNA, and a functional change
of ABCG2 by a change of a stabilization mechanism of the ABCG2
protein.
[0121] Examples of urate transport-related diseases and
inflammation-related diseases include hyperuricemia, gout,
rheumatoid arthritis, osteoarthritis, infertility, cerebral stroke,
an ischemic heart disease, arrhythmia (including atrial
fibrillation), photosensitivity, a chronic kidney disease and the
like. For example, infertility and photosensitivity were found in a
study of hyperuricemic pedigrees having a functional decline of
ABCG2. Also, it was confirmed that atrial fibrillation is found in
cases having a functional decline of ABCG2. These facts suggest
that these diseases may relate to a functional decline of
ABCG2.
[0122] Also, a higher serum uric acid level is apt to develop urate
transport-related diseases and inflammation-related diseases.
Accordingly, when the level is equal to or more than a given level
such as, for example, 8.0 mg/dl, it can be concluded that it is
highly possible the subject has a factor that is capable of
inducing urate transport failure, or a state or disease
attributable to that failure. The threshold level may be changed
suitably, for example, to 7 or 9.
[0123] The ABCG2 gene includes 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.
[0124] Determination of gene polymorphisms can be performed, 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
SmartAmp method, a Q-probe method (QP 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.
[0125] SNPs can be detected directly from a genomic DNA, for
example, by a direct sequencing method and the like. Also, a
particular genome DNA region may be amplified using a clone, or a
PCR method, an LCR method, an SDA method, an RCK method, a LAMP
method, a NASBA method and the like, and subsequently,
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.
[0126] 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. 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 a fluorescence, and
amplification of the template by the PCR reaction exponentially
enhances a fluorescence intensity. By labeling two allele-specific
probes with different fluorescent dyes, it is also possible to
distinguish between a homozygote and a heterozygote in one
assay.
[0127] 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 between 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 previously, it is possible to quantify
the flap released. By preparing two sets of flap-FRET probes and
labeling them by different fluorescent dyes, it is possible to
distinguish between a homozygote and a heterozygote in one
assay.
[0128] 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 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 samples in a short time.
[0129] The RCA method is a method in which a DNA-amplifying means
(a DNA polymerase moves on the template and synthesizes a long
complementary DNA using a circular single-stranded DNA as a
template) is applied to SNP typing. Identification of an SNP is
carried out by the presence or absence of amplification via the RCA
method. Thus, 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. In case 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.
[0130] The DNA chip method is a method in which hybridization with
a PCR-amplified, fluorescence-labeled cDNA or cRNA is carried out
using a DNA chip prepared by arranging oligonucleotide probes
containing a polymorphic site on a microarray. The method can
detect many SNPs rapidly.
[0131] Methods for determining polymorphisms in an amino acid
sequence include, for example, 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. 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 in a good
sensitivity. It is also possible to use a western blotting method
using an antibody against an ABCG2 polypeptide.
[0132] The MALDI-TOF/MS method which is one of mass analysis
methods is a method in which a protein 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,
ionization of the protein sample is carried out 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 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 is used, and an
analyzer of a triple quadrupole type using an electrospray
ionization method, of a hybrid type, or of an ion trap type and
other analyzers are also used.
[0133] The protein chip method can carry out comprehensively and
rapidly the interaction of a sample with proteins, peptides,
antibodies, expressed proteins and the like arranged on a basal
plate.
[0134] The evaluation kit according to the present invention is a
kit for evaluating whether or not the subject has a factor that is
capable of inducing urate transport failure, or a state or disease
attributable to that failure, the method including means for
detecting at least one SNP of V12M, R113X, Q126X, Q141K, F208S,
G268R, E334X, S441N, L447V, S486N, F506SfsX4, R575X, and C608X in
an ABCG2 gene, or a gene polymorphism having a relationship of
linkage disequilibrium with the SNP, using a sample containing
human genes of the subject.
[0135] Thus, the means may be provided as 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.
[0136] 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.
[0137] Also, polypeptides are those in which two or more amino
acids are linked by a peptide bond, and include relatively short
chain peptides or oligopeptides, and also long chain peptides
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.
[0138] The method for examining urate transport kinetics according
to the present invention uses nonhuman animals having a deficiency
of an ABCG2 gene, and includes a step for measuring their serum
uric acid levels. Also, the nonhuman animals having a deficiency of
an ABCG2 gene may be provided as means for examining the urate
transport kinetics.
[0139] Nonhuman animals include, for example, mammals such as
mouse, and also include tissues and cells constituting their body.
Also, 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. Similarly, nonhuman animals
overexpressing a human ABCG2 gene or a nonhuman ABCG2 gene,
nonhuman animals overexpressing a human ABCG2 gene or a nonhuman
ABCG2 gene containing at least one variation of V12M, R113X, Q126X,
Q141K, F208S, G268R, E334X, S441N, L447V, S486N, F506SfsX4, R575X,
and C608X, nonhuman cell lines or human cell lines having a
deficiency of an ABCG2 gene, nonhuman cell lines or human cell
lines overexpressing a human ABCG2 gene or a nonhuman ABCG2 gene,
nonhuman cell lines or human cell lines overexpressing a human
ABCG2 gene or a nonhuman ABCG2 gene containing at least one
variation of V12M, R113X, Q126X, Q141K, F208S, G268R, E334X, S441N,
L447V, S486N, F506SfsX4, R575X, and C608X, or cell membrane
vesicles prepared from these cell lines may be used.
[0140] The drug for urate transport-related diseases and
inflammation-related diseases according to the present invention is
a drug for reducing a factor that is capable of inducing urate
transport failure, or a state or disease attributable to that
failure, and contains a polynucleotide encoding an ABCG2 protein in
the form capable of introducing it into cells or a polypeptide
corresponding to an ABCG2 protein 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
conveniently improve the urate transport by administration via
injection and the like.
[0141] The form capable of introducing a polynucleotide into cells
means a form allowing introduction of the polynucleotide into cells
and expression of ABCG2 encoded so that an intracellular ABCG2 gene
expresses the ABCG2. 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 the ABCG2 in cells.
[0142] ABCG2 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. ABCG2 not having
any SNP of V12M, R113X, Q126X, Q141K, F208S, G268R, E334X, S441N,
L447V, S486N, F506SfsX4, R575X, and C608X, and ABCG2 not having at
least an SNP of Q126X are preferred. 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, Poxviridae, Adenoviridae, Picornaviridae and the
like.
[0143] 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.
[0144] 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 kidney-specific infection with a
virus vector is carried out, it is possible to introduce a
recombinant vector by inserting a catheter into an artery
transdermally and then inserting the catheter into a kidney artery
with checking the location of the catheter by X-rays.
[0145] An ABCG2 polypeptide can be prepared by a genetic
engineering technique using the above ABCG2 polynucleotide. Thus,
the ABCG2 polypeptide 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. In case the polynucleotide is integrated into an
expression vector, it is also possible to obtain the ABCG2
polypeptide 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.
[0146] Also, the ABCG2 polypeptide can be synthesized according to
a known chemical synthesis method.
[0147] The ABCG2 polypeptide 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.
[0148] 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. More specifically, the
peptide derivatives can be prepared in the form of a functional
group produced as a side chain on the peptide residues or as an
N-terminal group or a C-terminal group, in the range not destroying
any activity of an ABCG2 polypeptide 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.
[0149] Also, the ABCG2 polypeptide 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. Salts of a carboxyl group include, for example,
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, or procaine.
Acid addition salts include, for example, 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.
[0150] In order to formulate such an ABCG2 polypeptide in the form
capable of introducing it into cells, for example, use of a fused
polypeptide in which a cell membrane-permeating peptide is linked
to an N-terminal side of the polypeptide is mentioned. PTD of HIV-1
TAT or PTD of drosophila homeobox protein Antennapedia can be used
as the cell membrane-permeating peptide. The fused polypeptide can
be prepared by a genetic engineering technique, for example, using
a fused polynucleotide prepared by linking an ABCG2 polynucleotide
and a PTD polynucleotide. It is also possible to prepare a fused
polypeptide linked with a cell membrane-permeating 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 inserting a catheter into an
artery transdermally and then inserting the catheter into a kidney
artery while checking the location of the catheter by X-rays to
introduce a recombinant vector.
INDUSTRIAL APPLICABILITY
[0151] The present invention effectively evaluates whether or not
the subject has a factor that is capable of inducing urate
transport failure, or a state or disease attributable to that
failure, and therefore contributes to prevention and early
treatment of various urate transport-related diseases. Also, the
present invention contributes to treatment of urate
transport-related diseases without causing other undesirable
effects even after the onset. Accordingly, the present invention is
effective against inflammation-related diseases such as
hyperuricemia, gout, rheumatoid arthritis, osteoarthritis,
infertility, cerebral stroke, an ischemic heart disease, arrhythmia
(including atrial fibrillation), photosensitivity, and chronic
kidney disease, and also against 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, and therefore is industrially useful.
Sequence CWU 1
1
9019DNAHomo sapiens 1ttaagctga 929DNAHomo sapiens 2gaatatcaa
939DNAHomo sapiens 3caaatcaac 949DNAHomo sapiens 4gtggtacaa
959DNAHomo sapiens 5gactccaag 969DNAHomo sapiens 6cctgaaaag
979DNAHomo sapiens 7aatcagctg 989DNAHomo sapiens 8actttaaag
999DNAHomo sapiens 9atagctcag 9109DNAHomo sapiens 10ccagaacag
9119DNAHomo sapiens 11gctcttcat 9129DNAHomo sapiens 12tcatgttag
9139DNAHomo sapiens 13tttatgatg 9149DNAHomo sapiens 14ggatttacg
9159DNAHomo sapiens 15ctatgcaac 9169DNAHomo sapiens 16tatatccta
9179DNAHomo sapiens 17gtgagtaaa 9189DNAHomo sapiens 18gtatgtaca
9199DNAHomo sapiens 19gtgagtata 9209DNAHomo sapiens 20gtaagtatt
9219DNAHomo sapiens 21gtaatgtgg 9229DNAHomo sapiens 22gtaaatgct
9239DNAHomo sapiens 23gtatggttg 9249DNAHomo sapiens 24gtatatgaa
9259DNAHomo sapiens 25gtaaccagc 9269DNAHomo sapiens 26gtaagtaaa
9279DNAHomo sapiens 27gtgagtagg 9289DNAHomo sapiens 28gtaagtatg
9299DNAHomo sapiens 29gtgagtctg 9309DNAHomo sapiens 30gtatgtctt
9319DNAHomo sapiens 31gtaagtttt 9329DNAHomo sapiens 32tgtctgcag
9339DNAHomo sapiens 33tgtttacag 9349DNAHomo sapiens 34ctcttatag
9359DNAHomo sapiens 35tgccttaag 9369DNAHomo sapiens 36gtgatttag
9379DNAHomo sapiens 37ttaacttag 9389DNAHomo sapiens 38ctttcatag
9399DNAHomo sapiens 39attgcaaag 9409DNAHomo sapiens 40tttgaaaag
9419DNAHomo sapiens 41tcatggcag 9429DNAHomo sapiens 42gttctatag
9439DNAHomo sapiens 43ctgactaag 9449DNAHomo sapiens 44tttttgtag
9459DNAHomo sapiens 45gtgttatag 9469DNAHomo sapiens 46taatttcag
9479DNAHomo sapiens 47aaagataaa 9489DNAHomo sapiens 48tgggatcat
9499DNAHomo sapiens 49gttattaga 9509DNAHomo sapiens 50gatgatgtt
9519DNAHomo sapiens 51gttggaact 9529DNAHomo sapiens 52gatgtctaa
9539DNAHomo sapiens 53gttatcact 9549DNAHomo sapiens 54ccacagaga
9559DNAHomo sapiens 55atcattgtc 9569DNAHomo sapiens 56agcaggggt
9579DNAHomo sapiens 57acatgaata 9589DNAHomo sapiens 58gattgaagc
9599DNAHomo sapiens 59attttttca 9609DNAHomo sapiens 60gctttgcag
9619DNAHomo sapiens 61atgtactgg 962655PRTHomo sapiens 62Met 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
6321DNAHomo sapiensCDS(1)..(21) 63ttt atc cca gtg tca caa gga 21Phe
Ile Pro Val Ser Gln Gly 1 5 647PRTHomo sapiens 64Phe Ile Pro Val
Ser Gln Gly1 5 6521DNAHomo
sapiensCDS(1)..(21)misc_feature(10)..(12)The codon "rtg" codes for
Val or Met 65ttt atc cca rtg tca caa gga 21Phe Ile Pro Xaa Ser Gln
Gly 1 5 667PRTHomo sapiensMOD_RES(4)..(4)Val or Met 66Phe Ile Pro
Xaa Ser Gln Gly 1 5 6721DNAHomo sapiensCDS(1)..(21) 67ttt atc cca
atg tca caa gga 21Phe Ile Pro Met Ser Gln Gly 1 5 687PRTHomo
sapiens 68Phe Ile Pro Met Ser Gln Gly1 5 6926DNAHomo
sapiensCDS(1)..(15) 69ggt tac gtg gta caa gtaagtatta g 26Gly Tyr
Val Val Gln 1 5 705PRTHomo sapiens 70Gly Tyr Val Val Gln 1 5
7126DNAHomo sapiensCDS(1)..(15)misc_feature(13)..(15)The codon
"yaa" codes for Gln or a stop codon wherein the residue at this
position is absent 71ggt tac gtg gta yaa gtaagtatta g 26Gly Tyr Val
Val Gln 1 5 725PRTHomo sapiensMOD_RES(5)..(5)May or may not be
present 72Gly Tyr Val Val Gln 1 5 7326DNAHomo sapiensCDS(1)..(12)
73ggt tac gtg gta taagtaagta ttag 26Gly Tyr Val Val 1 744PRTHomo
sapiens 74Gly Tyr Val Val 1 7527DNAHomo sapiensCDS(1)..(27) 75aga
gaa aac tta cag ttc tca gca gct 27Arg Glu Asn Leu Gln Phe Ser Ala
Ala 1 5 769PRTHomo sapiens 76Arg Glu Asn Leu Gln Phe Ser Ala Ala 1
5 7727DNAHomo sapiensCDS(1)..(27)misc_feature(13)..(15)The codon
"mag" codes for Gln or Lys 77aga gaa aac tta mag ttc tca gca gct
27Arg Glu Asn Leu Xaa Phe Ser Ala Ala 1 5 789PRTHomo
sapiensMOD_RES(5)..(5)Gln or Lys 78Arg Glu Asn Leu Xaa Phe Ser Ala
Ala 1 5 7927DNAHomo sapiensCDS(1)..(27) 79aga gaa aac tta aag ttc
tca gca gct 27Arg Glu Asn Leu Lys Phe Ser Ala Ala 1 5 809PRTHomo
sapiens 80Arg Glu Asn Leu Lys Phe Ser Ala Ala 1 5 8127DNAHomo
sapiensCDS(1)..(27) 81cag tgt ttc agc agt gtt tca gcc gtg 27Gln Cys
Phe Ser Ser Val Ser Ala Val 1 5 829PRTHomo sapiens 82Gln Cys Phe
Ser Ser Val Ser Ala Val 1 5 8327DNAHomo
sapiensCDS(1)..(27)misc_feature(13)..(15)The codon "art" codes for
Ser or Asn 83cag tgt ttc agc art gtt tca gcc gtg 27Gln Cys Phe Ser
Xaa Val Ser Ala Val 1 5 849PRTHomo sapiensMOD_RES(5)..(5)Ser or Asn
84Gln Cys Phe Ser Xaa Val Ser Ala Val 1 5 8544DNAHomo
sapiensCDS(3)..(44) 85ga ttg aag cca aag gca gat gcc ttc ttc gtt
atg atg ttt acc 44 Leu Lys Pro Lys Ala Asp Ala Phe Phe Val Met Met
Phe Thr 1 5 10 8614PRTHomo sapiens 86Leu Lys Pro Lys Ala Asp Ala
Phe Phe Val Met Met Phe Thr 1 5 10 8744DNAHomo sapiensCDS(3)..(44)
87ga ttg aag cca aag gca gat gcc ttc ttc gtt atg atg ttt acc 44 Leu
Lys Pro Lys Ala Asp Ala Phe Phe Val Met Met Phe Thr 1 5 10
8814PRTHomo sapiens 88Leu Lys Pro Lys Ala Asp Ala Phe Phe Val Met
Met Phe Thr 1 5 10 8944DNAHomo sapiensCDS(3)..(32) 89ga ttg aag cca
aag gca gat gct tct tcg tta tgatgtttac cc 44 Leu Lys Pro Lys Ala
Asp Ala Ser Ser Leu 1 5 10 9010PRTHomo sapiens 90Leu Lys Pro Lys
Ala Asp Ala Ser Ser Leu 1 5 10
* * * * *