U.S. patent application number 17/409200 was filed with the patent office on 2022-02-24 for use of shroom3 in chronic kidney disease and chronic allograft nephropathy.
The applicant listed for this patent is Icahn School of Medicine at Mount Sinai. Invention is credited to John Cijiang He, Barbara Murphy.
Application Number | 20220056527 17/409200 |
Document ID | / |
Family ID | 1000005947490 |
Filed Date | 2022-02-24 |
United States Patent
Application |
20220056527 |
Kind Code |
A1 |
Murphy; Barbara ; et
al. |
February 24, 2022 |
USE OF SHROOM3 IN CHRONIC KIDNEY DISEASE AND CHRONIC ALLOGRAFT
NEPHROPATHY
Abstract
A method for identifying the risk of developing Chronic
Allograft Nephropathy (CAN) in a patient that received a kidney
transplant from a donor which comprises identifying the race of the
donor; determining the levels of SHROOM 3 expression in a kidney
biopsy specimen obtained from the patient at a predetermined time
after transplant; comparing the level of SHROOM 3 expression in the
biopsy specimen with the levels of SHROOM 3 expression in a
control; determining if the level of SHROOM 3 expression in the
allograft is significantly higher than in the control, and
diagnosing the patient as being at risk for CAN if the level of
SHROOM 3 expression in the specimen is significantly higher than in
the control.
Inventors: |
Murphy; Barbara; (Pelham
Manor, NY) ; He; John Cijiang; (Forest Hills,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Icahn School of Medicine at Mount Sinai |
New York |
NY |
US |
|
|
Family ID: |
1000005947490 |
Appl. No.: |
17/409200 |
Filed: |
August 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14773687 |
Sep 8, 2015 |
11098361 |
|
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PCT/US14/22607 |
Mar 10, 2014 |
|
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17409200 |
|
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61777328 |
Mar 12, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/347 20130101;
C12Q 2600/136 20130101; C12Q 2600/118 20130101; G01N 2800/50
20130101; C12Q 2600/158 20130101; G01N 2500/10 20130101; G01N
2800/245 20130101; C12Q 2600/156 20130101; C12Q 1/6883
20130101 |
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883 |
Goverment Interests
GOVERNMENT CLAUSE
[0002] This invention was made with government support under
1U01AI070107-01 awarded by The National Institutes of Health. The
government has certain rights in the invention.
Claims
1.-17. (canceled)
18. A method for determining if a Caucasian allograft recipient
that received a kidney allograft from a Caucasian kidney donor is
at risk for allograft rejection which comprises the steps of
determining the levels of SHROOM 3 expression in a kidney biopsy
specimen obtained from the allograft recipient at a predetermined
time after transplant; comparing the level of SHROOM 3 expression
in the biopsy specimen with the levels of SHROOM 3 expression in
control biopsies obtained from normal subjects; wherein if the
level of SHROOM 3 expression in the allograft is higher than the
expression levels of SHROOM 3 in a control the allograft recipient
is at risk for allograft rejection.
19. The method of claim 18 comprising administering an
immunosuppressive agent to the allograft recipient at risk for
allograft rejection.
20. The method of claim 19 wherein said immunosuppressive agent is
cyclosporin.
21. The method of claim 18 which comprises administering an
angiotensin converting enzyme inhibitor to said allograft
recipient.
22. The method of claim 18 wherein said levels of Shroom3
expression are determined by Real Time Polymerase Chain Reaction
(RT-PCR).
23. The method of claim 18 wherein said allograft recipient at risk
for allograft rejection is suffering from a disease selected from
the group consisting of Chronic Allograft Nephropathy (CAN),
Chronic Kidney Disease (CKD), obstructive uropathy, HIV-associated
nephropathy, diabetic nephropathy and fibrosis.
24. A kit for identifying renal allograft recipients at risk for
developing CAN or CKD selected from the group consisting of
obstructive uropathy, HIV-associated nephropathy and diabetic
nephropathy comprising in separate containers primers for RT-PCR
SHROOM3 expression assays, a microarray for gene expression
analysis and primers for RT-PCR analysis of the rs17319721 risk
allele, buffers and instructions for use.
25. A method for identifying the risk of developing fibrosis in a
Caucasian kidney allograft recipient after receiving a kidney
transplant from a Caucasian donor, comprising the steps of:
determining if said donor expresses the rs 17319721 SNP risk
allele; conducting a genetic analysis to determine if said donor is
homozygous for said risk allele; wherein if said donor is
homozygous for said risk allele then said allograft recipient is at
risk for fibrosis.
26. The method of claim 18 which comprises treating the allograft
recipient determined to be at risk for allograft rejection with an
anti-rejection agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional, and claims priority to
U.S. application Ser. No. 14/773,687, filed Sep. 8, 2015, now U.S.
Pat. No. 11,098,361, issued Aug. 24, 2021, which is the U.S.
National Phase Application under 35 U.S.C. .sctn. 371 of
International Patent Application No. PCT/US2014/022607 filed Mar.
10, 2014, which claims the benefit of U.S. Provisional Application
No. 61/777,328 filed Mar. 21, 2013, all of which are incorporated
by reference herein. The International Application was published in
English on Oct. 2, 2014 as WO2014/159227 A1 under PCT Article
21(3).
SEQUENCE LISTING
[0003] This application contains a Sequence Listing that has been
submitted electronically as an ASCII text file named
27527-0124002SEQ.txt. The ASCII text file, created on Sep. 21,
2021, is (8,192 bytes in size. The material in the ASCII text file
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0004] The present invention pertains to methods to identify
patients suffering from kidney diseases and predict the development
and progression of kidney fibrosis in renal allograft recipients.
The invention includes a kit for identifying patients suffering
from kidney diseases and for predicting the development and
progression of kidney diseases and tubulo-interstitial fibrosis in
renal allograft recipients.
BACKGROUND OF THE INVENTION
[0005] Chronic Kidney disease (CKD) affects 10% of US adults with
rising incidence and prevalence worldwide {Coresh J, 2007}.
End-stage renal disease (ESRD) requires renal replacement therapy
(RRT) and currently affects over 500,000 US adults. In addition to
conferring risk for end stage renal disease (ESRD), CKD increases
the risk of cardiovascular disease and all-cause mortality {Weiner
D, 2004}. Tubulo-interstitial fibrosis (TIF) is a final common
pathogenic process for CKD from varied etiologies leading to the
development of ESRD. TIF is also a primary component of chronic
allograft nephropathy (CAN) and is associated with progressive
decline of estimated glomerular filtration rate (eGFR) in the renal
allograft {Chapman J, 2005} {Nankivell B J, 2003}. CAN represents
the most common cause of death-censored long-term graft loss and is
measured histologically by the chronic allograft dysfunction index
score (CADI score) {Isoniemi H, 1992} {Yilmaz S, 2007}. To date,
there is no effective anti-fibrotic therapy to prevent the
progression of CKD or CAN. Presently, some patients with CKD or CAN
will eventually progress to ESRD and need RRT. For instance, renal
allograft recipients represented 13.5% of patients on the kidney
transplant waitlist and 15% of all transplants performed in 2005
{Magee J C, 2007}. Renal allograft recipients have a 30%
probability of requiring RRT or re-transplantation at 10-years
{USRDS 2012}. RRT and ESRD-care are a disproportionate and
burgeoning financial burden to Medicare {Iglehart, 2011}. It is
therefore critical to identify new sensitive biomarkers to predict
the development of kidney fibrosis. Furthermore, these markers
could represent targets for therapeutic intervention to prevent the
development of TIF at an early stage, thereby preventing
progression to ESRD.
[0006] Allograft biopsy based studies (both for-cause and protocol)
have provided insight into how early allograft changes correlate
with long term allograft outcomes {Gago M, 2012} {Rush D, 1994}
{Seron D, 1997} {Cosio F, 2005} {Park W, 2010}. More recently,
distinct biopsy and blood gene-expression profile (transcriptome)
signatures have been shown to classify patients with acute
rejection, chronic rejection, those on immunosuppression, and
operationally tolerant recipients {Akalin E, 2010} {Akalin E, 2001}
{Flechner S, 2004} {Donauer J, 2003} {Sarwal M, 2003} {Reeve J,
2009} {Scherer A, 2003} {Sagoo P, 2010} {Newell K, 2010}. These
gene panels have been able to improve upon histological classifiers
alone {Sarwal M, 2003} {Reeve J, 2009}. Genome-wide association
studies have also strongly linked a single-nucleotide polymorphism
(SNP) in the Shroom3 gene (rs17319721) with incident and prevalent
CKD by eGFR in population-based cohorts of European ancestry
{Kottgen A, 2009} {Boger C, 2011}. The Shroom3 gene encodes a PDZ
domain-containing protein that can directly bind F-actin and
regulate its subcellular distribution in cells. Complete absence of
or defective Shroom3 causes open neural tube defects and neonatal
death in mice {Hildebrand J, 1999}. In MDCK kidney cell lines,
Shroom3 localizes at the apical and junctional complexes and is
critical to the maintenance of normal epithelial cell phenotype
{Hildebrand J, 2005}. It is also known that C-terminal domain of
Shroom3 interacts with Rho-Kinases (ROCKs) to facilitate myosin
phosphorylation and actin contraction {Nishimura T, 2008}. However,
whether Shroom3 plays a role in kidney fibrosis in CKD or CAN is
yet unknown.
[0007] What are needed in the art are markers whose expression can
be used to identify patients suffering from kidney diseases and
predict the development of kidney fibrosis. In addition, such
markers are needed to identify renal allograft recipients who are
at risk for developing CAN and represent targets for therapeutic
intervention to prevent the development of TIF at an early stage,
thereby preventing progression to ESRD.
SUMMARY OF THE INVENTION
[0008] Shroom3 is a novel candidate gene whose expression in a
renal allograft precedes and predicts decreased renal function and
TIF. Higher allograft Shroom3 levels predict histological
progression of CAN. It has also been found that these relationships
correlate best in recipients of white-donor kidneys. The findings
confirm for the first time that the previously described chronic
kidney disease (CKD)-associated Shroom3 locus (rs17319721) {Kottgen
A, 2009} {Boger C, 2011} mediates its effect through increased
Shroom3 expression. In addition, it has been discovered that
Shroom3 has a salutary role in canonical TGF-beta signaling and
Collagen-1 production.
[0009] In one aspect, the present invention provides method for
identifying the risk of developing Chronic Allograft Nephropathy
(CAN) in a patient that received a kidney transplant from a donor
which comprises identifying the race of the kidney donor;
determining the levels of SHROOM3 expression in a kidney biopsy
specimen obtained from the patient at a predetermined time after
transplant; comparing the level of SHROOM3 expression in the biopsy
specimen with the levels of SHROOM expression in a control;
determining if the level of SHROOM3 expression in the allograft is
significantly higher than in the control, and diagnosing the
patient as being at risk for CAN if the level of SHROOM3 expression
in the specimen is significantly higher than in the control.
[0010] In a further aspect the present invention provides a method
for identifying the risk of developing Chronic Allograft
Nephropathy (CAN) in a patient that received a kidney from a donor
comprising the steps of identifying the race of the kidney donor;
obtaining a renal allograft biopsy sample from the patient;
determining the levels of SHROOM3 expression in said biopsy;
comparing the levels of SHROOM3 expression in said biopsy with the
levels of SHROOM3 expression in a control and advising the patient
as being at risk of developing CAN if the levels of SHROOM3 in the
sample are significantly higher than in the control and the kidney
donor is Caucasian.
[0011] In yet a further aspect, the present invention provides a
method for identifying the risk of developing a renal disease in a
Caucasian patient that received a kidney from a donor, comprising
the steps of determining if said donor expresses the rs 17319721
SNP risk allele and conducting a genetic analysis to determine if
said donor is homozygous for said risk allele, wherein if said
donor is homozygous for said risk allele then said patient is at
risk for developing a renal disease.
[0012] In yet a still further aspect, the present invention
provides a method for identifying the risk of developing fibrosis
in a Caucasian kidney donor, comprising the steps of determining if
said donor expresses the rs 17319721 SNP risk allele, and
conducting a genetic analysis to determine if said donor is
homozygous for said risk allele; wherein if said donor is
homozygous for said risk allele then said donor is at risk for
fibrosis.
[0013] In a still further aspect the present invention provides a
method for identifying the risk of developing a progressive kidney
disease selected from the group consisting of Chronic Allograft
Nephropathy (CAN) and Chronic Kidney Disease (CKD) of a Caucasian
patient which comprises: determining the levels of SHROOM3
expression in a kidney biopsy specimen obtained from the patient at
a predetermined time, comparing the level of SHROOM3 expression in
the biopsy specimen with the levels of SHROOM3 expression in a
control, determining if the level of SHROOM3 expression in the
specimen is significantly higher than in the control, and
diagnosing the patient as being at risk for said disease if the
level of SHROOM3 expression in the specimen is significantly higher
than in the control.
[0014] In a still further aspect, the present invention provides a
kit for identifying patients suffering from a renal disease and at
risk for developing CAN or CKD comprising in separate containers
primers for use in RT-PCR assays for SHROOM3 expression, RT-PCR for
detecting the rs 17319721 SNP risk allele, a positive control,
buffers and instructions for use.
[0015] These and other aspects of the present invention will be
apparent to those of ordinary skill in the art in light of the
present description, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: a diagram depicting the number of participants in
the study reported herein, and showing that 160 participants had
3-month allograft biopsy RNA extracted for microarray. Among these
160 patients, at 12-months post-transplant, 147 had
eGFR-creatinine, 101 had CADI-12 and 85 had CADI-3 and -12
reported.
[0017] FIG. 2A-2D: graphs depicting (2A) the correlation of
allograft log Shroom3 expression (microarray) at 3 months and eGFR
creatinine at 12 months (r=-0.20; p=0.007); (2B) the correlation of
allograft log Shroom3 expression (microarray) at 3 months and
CADI-12 months (Pearson R-0.22; p=0.027; (2C) Shroom3 expression
was higher in all donors with progressive fibrosis (Delta CADI=2 or
more; n=17) vs those without significant progression (Delta
CADI<2; n=68); and (2D) the correlation between 3-month Shroom3
expression was strongest in deceased-donor kidneys and 12-month
CADI (r=0.34; p=0.0088) [Line represents mean; Whiskers=SD].
[0018] FIG. 3A-3D: graphs depicting (3A) the correlation of Fold
change Shroom3 expression (RT-PCR) at 3 months and CADI-12 months
(Pearson r=0.4169; p=0.0103); (3B) the correlation of Fold change
Shroom3 expression (RT-PCR) at 3 months and eGFR creatinine at 12
months (r=0.3230; p=(3C) the correlation between allograft log
Shroom3 expression (microarray) at 3 months and Fold change Shroom3
expression at 3 months (RT PCR) (Pearson r=0.5613; p=0.0008); and
(3D) the regression line of relationship between log Shroom3
expression at 3 months and simultaneous CADI score--No relationship
could be identified
[0019] FIG. 4A-4C: graphs depicting (4A) the correlation of
allograft log Shroom3 expression at 3 months and CADI-12 months in
recipients of white-donor kidneys (r=0.2538; p=0.02); (4B) the
correlation of allograft log Shroom3 expression to eGFR-creatinine
in recipients of white-donor kidneys (r=-0.25; p=0.008); and (4C)
Log Shroom3 expression was higher in WDKRs with progression of
fibrosis (Delta CADI.gtoreq.2; n=14) vs than those without
significant progression (Delta CADI<2; n=56) [*p<0.05;
**p<0.001; ***p<0.0001]
[0020] FIG. 5A-5C: graphs depicting that (5A) there is no
correlation between allograft log Shroom3 expression (microarray)
at 3 months and 12-month CADI in living donor recipients (LDRs)
and: (5B) in non-WDKRs; (5C) No correlation between 3-month Shroom3
and eGFR-12 m in non-WDKRs.
[0021] FIG. 6A-6B: graphs illustrating that (6A) Shroom3 SNP
(rs17319721) is differently distributed between whites and
non-whites (amongst both donors and recipients). Whites have a
higher prevalence of effect allele (p<0.0001) (6B): Homozygosity
for the risk allele in donor kidneys is associated with
significantly increased allograft Shroom3 expression (p=0.033)
whiskers: 5th-95th percentile; Line at Median; p=0.0183)
[*p<0.05; **p<0.001; ***p<0.0001]
[0022] FIG. 7A-7C: graphs depicting that (7A, 7B, respectively)
Shroom3 expression was increased in both WDKRs and non-WDKRs with
the presence of effect-allele (A) in the donor and (7C). Shroom3
expression was not significantly affected Recipient SNP type
(whiskers: Min-Max; Line at Median; p=0.0309) [*p<0.05;
**p<0.001; ***p<0.0001]
[0023] FIG. 8A-8B: graphs that illustrate (8A) Allele prevalence of
the Shroom3 SNP in 354 white recipients (as %) according to ESRD
etiology. Recipients with ESRD from Diabetes (49%) had the
significantly greater risk-allele prevalence compared to unrelated
donors. (8B): Allele prevalence of the Shroom3 SNP in 3247 patients
of the study. Risk allele prevalence was highest in Diabetics with
CKD (51%) [*p<0.05; **p<0.001; ***p<0.0001].
[0024] FIG. 9: a construct map of Luciferase-reporter plasmids used
in Example 5 below.
[0025] FIG. 10A-10B: FIG. 10A is a graph that depicts that
Luciferase-reporter plasmids with A-allele enhancer element showed
greater activity than G-allele and promoter-only plasmids; FIG. 10B
is a Western Blot that illustrates that Nucleoprotein extracted
from 293-T cells showed enhanced binding to oligonucleotide
sequences containing the G-allele.
[0026] FIG. 11A-11B: FIG. 11A is a graph illustrating that in
PRCEC, TGF-.beta. treatment increases Shroom3 in a dose-dependent
(up to 5 ng/ml) and time dependent fashion by RT-PCR (error bars:
mean in PRCEC, TGF-.beta. treatment increases Shroom3 in a time
dependent fashion (up to 5 ng/ml) (error bars: mean.+-.SEM)
[*p<0.05; **p<0.001; ***p<0.0001]. FIG. 11B is a Western
Blot that illustrates that TGF-.beta. treatment increases Shroom3
in a time-dependent fashion.
[0027] FIG. 12A-12B: FIG. 12A is a graph that depicts the results
of Example 6 that shows TGF-beta increases Shroom3 expression in a
beta-Catenin/TCF7l2 dependent manner. Quercetin (beta-Catenin
inhibitor) and BC-21 (TCF7L2 inhibitor) inhibited the increase in
Shroom3 expression induced by TGF-beta (RT-PCR). 12B is a Western
blot that depictsShroom3 protein increases in a
Beta-Catenin/TGF-.beta. dependent manner.
[0028] FIG. 13A-13B: FIG. 13A is a chart depicting Shroom3
overexpression in PRCEC with PC-SHROOM3 transfection confirmed by
RT-PCR and FIG. 13B by Western blot respectively (mean.+-.SEM)
[*p<0.05; **p<0.001; ***p<0.0001.
[0029] FIG. 14A-14C: FIG. 14A is a graph depicting the results of
Example 7 and showing the effect on TGF-beta induced expression of
profibrotic matrix markers by RT-PCR. Shroom3 over-expression alone
increased Collagen-1 and Fibronectin production while knockdown
suppressed these matrix markers (mean.+-.SEM) [*p<0.05;
**p<0.001; ***p<0.0001]; FIG. 14B is a Western blot showing
Shroom3 over-expression enhanced Phosphorylation of Smad-3 in
response to TGF-beta in PRCEC at 15 minutes (Right); FIG. 14C is a
photograph showing that TGF-.beta.1 treatment of
SHROOM3-overexpressed PRCEC also developed more prominent F-actin
bundles compared to TGF.beta.1-treated cells without SHROOM3
overexpression (FIG. 14C--Upper and lower Panels).
[0030] FIG. 15A-15B: Graphs that depict the results of Example 7
which establishes that Shroom3 overexpresses in PRCEC with
PC-SHROOM3 transfection, confirmed by RT-PCR (FIG. 15A) and Western
blot (FIG. 15B) respectively (mean.+-.SEM) [*p<0.05;
**p<0.001; ***p<0.0001]
[0031] FIG. 16A-16D: (16A) Representative genotyping results for
ROSA-RTTA (upper) and COLTGM (lower) are depicted. (16B) Western
blots of phospho-SMAD3 (p-SMAD3), total SMAD3 (SMAD3), Shroom3 and
.beta.-actin from mouse kidney cortex lysates from UUO kidneys of
non-Dox-fed vs Dox-fed mice are displayed (n=2). (16C) Left to
right--Photographs showing representative, low power (10.times.)
images from Control kidney and UUO Kidney of non-Dox-fed animals,
and UUO kidneys of Dox-fed animals are displayed. Upper
row--Periodic acid Schiff stain, middle row--Picrosirius red stain.
Lower row--immunolabeling for COL1A1 (TRITC-Alexa fluor) was
performed on snap frozen kidney cortex sections. Representative
images are displayed. Graph represents morphometric quantification
of Area COL1A1 staining/total area (%) in 5 random high power
fields per animal. (16D) Bar graphs depicting SHROOM3 and COL1A1
mRNA expression by RT PCR in control and UUO kidneys of DOX- and
non-DOX fed animals (normalized to GAPDH; n=5; mean.+-.SEM; ANOVA
with post-test Bonferroni comparison; *P<0.05).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0032] The term "about" or "approximately" usually means within an
acceptable error range for the type of value and method of
measurement. For example, it can mean within 20%, more preferably
within 10%, and most preferably still within 5% of a given value or
range. Alternatively, especially in biological systems, the term
"about" means within about a log (i.e., an order of magnitude)
preferably within a factor of two of a given value.
[0033] The term "significantly higher levels of Shroom3 expression"
is defined herein as between about 1.4 and about 5-fold higher than
in the control.
[0034] The present invention is based on the discovery that the
levels of SHROOM3 expression are significantly higher in kidney
allograft recipients that are at risk for developing CAN when
compared to control biopsies obtained normal subjects. Pursuant to
the present invention, SHROOM3 expression levels are determined
from biopsies obtained from kidney allograft recipients and
compared to biopsies obtained from controls which are normal kidney
samples such as living donor baseline biopsy samples or kidney
samples from nephrectomy surgeries using techniques well known in
the art such as immunostaining but preferably by Real Time
Polymerase Chain Reaction (RT-PCR).
[0035] In a Genomics of Chronic allograft rejection study protocol,
biopsies were obtained from all enrolled patients at different time
points (See Examples below). From the DNA-microarray performed on
allograft biopsies at 3-months, genes whose differential expression
correlated with CADI-score and renal function at 12-months were
identified and ranked. Among the top ranked genes in this list was
SHROOM3 (Unpublished data).
[0036] Genome-wide association studies have also strongly linked a
single-nucleotide polymorphism (SNP) in the SHROOM3 gene
(rs17319721) with incident and prevalent CKD by eGFR in
population-based cohorts of European ancestry {Kottgen A, 2009}
{Boger C, 2011}. The SHROOM3 gene encodes a PDZ domain-containing
protein that can directly bind F-actin and regulate its subcellular
distribution in cells. Complete absence of, or defective SHROOM3
causes open neural tube defects and neonatal death in mice
{Hildebrand J, 1999}. In MDCK kidney cell lines, SHROOM3 localizes
at the apical and junctional complexes and is critical to the
maintenance of normal epithelial cell phenotype {Hildebrand J,
2005}. It is also known that the C-terminal domain of SHROOM3
interacts with Rho-Kinases (ROCKs) to facilitate myosin
phosphorylation and actin contraction {Nishimura T, 2008}.
[0037] In the study reported herein, 589 patients were enrolled.
Allograft biopsies were obtained at 0, 3, 12, and 24 months
post-transplant with Chronic allograft dysfunction index score
(CADI) reported from a core lab. Gene expression microarray
analysis was performed on 3 month biopsies (Affymetrix: human
exon-1 chip) and correlation to 12-month CADI and eGFR were
analyzed (n=160). Overexpression and lentiviral suppression studies
were performed on human primary tubular cells (RPTE). SHROOM3
gene-expression was found to correlate linearly with fibrosis and
negatively with eGFR at 1 year (n=101; p<0.05). This was
confirmed by RT-PCR independently (n=36). No correlation was seen
between SHROOM3 expression and 3 month CADI (n=137).
[0038] A SNP in SHROOM3 (rs17319721) has been linked to CKD in
genome-wide association studies. As disclosed herein, it has been
found that the presence of at least 1 copy of the risk allele (i.
e. A/G or A/A) in a donor's DNA is associated with higher
intragraft SHROOM3 expression at 3 months (n=136; p=0.02). The risk
allele was more prevalent in white vs. non-white donors. As shown
in Example 4 below, the SNP risk allele is more frequent in white
diabetics with renal disease. Therefore, the present invention
provides a method for identifying the risk of developing CAN in a
diabetic Caucasian patient suffering from renal disease that
received a kidney from a donor, comprising the steps of obtaining a
blood sample from the donor at baseline and conducting a genetic
analysis to determine if the donor is homozygous for the risk
allele. As shown in Example 9 below, the method can be practiced
with non-diabetic Caucasian patients. If the donor is homozygous
for the risk allele then the patient is at risk for CKD or CAN.
This method can only be performed if the donor is available, which
is not always the case. If the donor is not available then SHROOM3
expression levels in the patient can be examined. While in
recipients of white-donor kidneys SHROOM3 expression was predictive
of CADI at 12 months, this was not true for non-white or living
donor recipients.
[0039] Overexpression of SHROOM3 in RPTE increased, while
lentiviral suppression markedly diminished type-1 collagen
production (p<0.01). SHROOM3 excess facilitated, while
suppression inhibited canonical TGF-beta signaling, evidenced by
Phospho-Smad3 and profibrotic marker production. Further, in FVB/N
mice with CKD (HIV-nephropathy and Unilateral Ureteric
Obstruction), SHROOM3 expression was increased compared to controls
as determined by RT-PCR. Therefore, the present invention is not
limited to CAN but is useful for other CKD such as obstructive
uropathy, HIV-associated nephropathy and diabetic nephropathy.
[0040] Without wishing to be bound by theory, it is believed that
Shroom3 is involved in any disease that involves fibrosis such as
liver fibrosis and lung fibrosis. Therefore, the materials and
methods described herein will be useful for monitoring the
progression of such diseases. As shown below in Example 10 renal
interstitial fibrosis was significantly abrogated in Shroom3
knockdown animals in a mouse model. These results validate the role
of SHROOM3 in these diseases.
[0041] The present invention also provides kits for use in the
methods disclosed herein. The kits comprise, in separate
containers, the following components: primers for RT-PCR SHROOM3
expression assays, a microarray for gene expression analysis and
primers for RT-PCR analysis of the rs17319721 risk allele, buffers
and instructions for use. A non-limiting list of primers for use in
the Kits is set forth in Table 5. The positive control comprises
the cells over-expressing SHROOM3 or brain tissues, such as
neuroepithelium, which are known to have high levels of SHROOM3
expression. (Shroom3-mediated recruitment of Rho kinases to the
apical cell junctions regulates epithelial and neuroepithelial
planar remodeling--Tamako Nishimura and Masatoshi Takeichi,
Development 135, 1493-1502 (2008) doi:10.1242/dev.019646).
[0042] The present invention is directed to methods for identifying
kidney allograft recipients who are at risk for developing kidney
diseases such as CAN or CKD. When such patients are identified, the
present invention includes methods for treating such patients. Such
methods include, without limitation, administration of
immunosuppressive drugs, i.e. a calcineurin inhibitor (CNI), such
as cyclosporine or tacrolimus, or a less fibrogenic
immunosuppressive drug such as mycophenolate mofetil (MMF) or
sirolimus. Since patients who are identified as being at risk for
developing CAN or CKD have impaired renal function and often suffer
from hypertension, administration of an angiotensin converting
enzyme inhibitor (ACEI) such as lisinopril or angiotensin II
receptor blockade such as losartan, to such patients is within the
scope of the present invention.
[0043] The present invention also provides methods using SHROOM3 as
a target to screen for drugs useful for the treatment of CAN and
CKD. The 293T cells transfected with the SHROOM3
A-allele/luciferase construct described in Example 5 below can be
used in screening assays to identify drugs for the treatment of
fibrotic diseases mediated by SHROOM3. The cells can be seeded in
96 well microplates, contacted with drug candidates and assayed for
changes in luciferase activity. SHROOM3 activity (i.e., luciferase)
at baseline will be measured, and inhibitors Quercetin and/or
BC-21, can be used as positive controls.
[0044] As an alternative, the 293T cells will be treated with TGFB1
(5-10 ng/ml), a known up-regulator of SHROOM3 expression and these
cells (with a higher baseline of SHROOM3 expression) are used in
the screening assays. These assays can be developed in a high
throughput format.
[0045] Primers for use in RT-PCR assays for the A allele and for
generating luciferase reporter constructs are set forth in Table
5.
[0046] In summary, pursuant to the present invention, SHROOM3 has
been identified as a novel candidate gene whose expression in the
renal allograft precedes and predicts the derangement of renal
function and TIF in CAN. Significantly higher allograft SHROOM3
levels can be used to predict histological progression of CAN. In
addition, it has been discovered that these relationships are most
predictive in recipients of white-donor kidneys. These findings
confirm for the first time that the previously described
CKD-associated SHROOM3 locus (rs17319721) {Kottgen A, 2009} {Boger
C, 2011} mediates its effect through increased SHROOM3 expression.
Finally, the in vitro studies described herein suggest a salutary
role for SHROOM3 in canonical TGF-beta signaling and Collagen-1
production in renal tubular cells. Taken together, these findings
demonstrate that SHROOM3 is a therapeutic target in both CAN and
CKD and suppression of its level can be used to inhibit the
progression of TIF and retard ESRD.
[0047] The present invention is described below in working examples
which are intended to further describe the invention without
limiting the scope thereof.
[0048] In the Examples below, the following Materials and Methods
were used.
[0049] Biopsies and RNA extraction: Real-time, ultrasound-guided,
renal allograft biopsies were obtained at 0, 3, 12, and 24 months
post-transplant, at 3 of 5 clinical sites. Two Cores were extracted
using 18 G spring-loaded biopsy needles when possible. If a single
core was obtained, preference was given to RNA extraction
(QIAGEN-ALLprep kit, Valencia, Calif. USA) at the 3-month visit and
to histological analysis at 12-months. Tissue for gene-expression
studies was stored immediately in RNA-later and shipped at
-20.degree. C. to a genomics core facility.
[0050] Reverse transcription: Extracted biopsy RNA were reverse
transcribed using Sensiscript single-step RT (Qiagen) and Oligo-DT
primer (Qiagen) with starting total-RNA amount of 55 ng. Extraction
samples with RNA concentrations <5 ng/mcl by nanodrop were not
used. For in vitro studies we used Superscript-III (Invitrogen-Life
technologies, Grand Island, N.Y.) with starting total RNA 500-1000
ng.
[0051] RT-PCR: Intron-spanning primer sets were designed for
Shroom3 using Primer-BLAST (NCBI) and PCR amplicons were confirmed
by both melting curve analysis and agarose gel electrophoresis.
Shroom3 expression was assayed in an internal and external cohort
of patients by real-time polymerase chain reaction (RT-PCR)
(Applied biosystems 7500 cycler).
[0052] Shroom3 SNP analysis: Targeted genotyping was performed for
Shroom3 SNP (rs17319721) using Taqman SNP analysis assay (Cat No:
4351379 Applied Biosystems, Foster City, Calif.). DNA was extracted
(QIAGEN-ALLprep kit, Valencia, Calif. USA) from pre-implantation
biopsies or blood for donor SNP and from peripheral blood for
recipient SNP assay. Automated analysis using Genotyping software
from Applied Biosystems was performed (Applied Biosystems 7500
cycler).
[0053] Shroom3 promoter-enhancer constructs and Luciferase-reporter
assay: Promoter fragments spanning -3000 bases 5' of the
translation start site of SHROOM3 were PCR amplified using primer
sets that optionally included twenty-four base-pair sequences of
the intron-1 of SHROOM3 including the rs17319721 site containing
either of the two alleles (A or 1. G) (Table-1). Restriction sites
for KpnI were introduced in all forward primer and Hind III in the
reverse ones. The PCR products were then cloned into luciferase
reporter vector-pGL3 Basic (E-1751, Promega, WI, USA) using Kpn I
and Hind III sites. This generated 3 reporter plasmids--Promoter
only, Promoter with either intronic-A or -G sequences.
[0054] Transient transfection of these constructs (1 mcg each) with
Renilla luciferase reporter plasmid-pTL-TK (200 ng) was carried out
in HEK293T-cells plated on 6-well plates at 80% confluence (Polyjet
reagent, SignaGen labs, Rockville, Md.). After 24-hours, cells were
lysed and protein extracted. Renilla and Luciferase activity was
measured in lysates using dual luciferase assay system (PJK
Germany) on microplate reader according to manufacturer protocol.
Results were expressed and Luciferase: Renilla ratio.
[0055] Western Blotting: Cells were lysed with a buffer containing
1% Triton, a protease inhibitor mixture and tyrosine and
serine/threonine phosphorylation and phosphatase inhibitors.
Lysates were subjected to immunoblot analysis using Rabbit
anti-Shroom3 (a gift from Dr Jeffrey Hildebrand, Pittsburgh),
anti-V5 tag antibody (R960-25, Invitrogen-Life technologies),
Phospho-Smad3 antibody (Rabbit polyclonal-pS423/425) and Smad-3
(Rabbit monoclonal Smad-3, #9523, from Epitomics, Burlingame,
Calif.). Densitometry was performed as previously described
{Gassman 2009}.
[0056] Overexpression studies: A human Shroom3 construct (Open
Biosystems, Lafayette, Colo.) was cloned into mammalian expression
vector PC-DEST40 (Invitrogen, Carlsbad, Calif.) with C-terminal V5
and Histidine tags using Clonase-II recombinase (Invitrogen).
Electroporative transfection using Lonza Nucleofector Technology
(Basic Nucleofector kit for Primary Mammalian Epithelial Cells,
Program T20) was used to transfect primary renal cortical
epithelial cells (PRCEC) as described previously {Jin Y, 2012}.
Forced expression was confirmed in PRCEC by RT-PCR and Western blot
with empty destination vector transfection as control. Profibrotic
extracellular matrix markers were analyzed in PRCEC by Real-time
polymerase chain reaction (RT-PCR). Effect of TGF-.beta. treatment
on matrix marker production in Shroom3-transfected cells was
assayed in nutrient starved medium 36 hours after transfection.
Phosphorylation of Smad-3 was measured at 5, 15 and 30 minutes of
treatment with TGF-Beta.
[0057] Shroom3 SIRNA suppression studies: Human Shroom3 short
hairpin clones (Open Biosystems, Lafayette, Colo.) were tested for
Shroom3 suppression by RT-PCR and Western blot in 293-T cells. The
selected GFP-tagged hairpin was transfected into 293-T cells along
with envelope plasmids (Polyjet reagent) to generate a mammalian
VSV pseudotyped lentiviral expression construct. PRCEC were
infected using this lentiviral construct and Shroom3 suppression
was confirmed. For in vivo Shroom3 suppression in mouse, potent
suppressive hairpin sequences were shortlisted using a sensor
Ping-Pong assay capable of deciphering elaborate shRNA libraries
(Mirimus Inc., Long Island, N.Y.). Mouse podocytes were transduced
with these sequences on a Mir30 lentiviral backbone and tested for
Shroom3 suppression using RT-PCR and Western blot. Two sequences
were selected for embryonic stem cell injection into mice to
develop a Tetracycline-inducible Shroom3-ShRNA mouse model.
[0058] Characteristics of the Participants
[0059] Five hundred eighty nine recipients were enrolled in the
study from 5 centers at the completion of enrollment for the study.
The demographic and clinical characteristics of donors and
recipients in the cohort are listed in Table-1. Demographics and
clinical characteristics of patients included in microarray studies
are detailed in Table-2.
Example 1
[0060] SHROOM3 is Upregulated and Associated with Progression of
CAN
[0061] To identify genes that could potentially contribute to the
development of CAN we performed an interim analysis for the first
66 subjects of the cohort who had the gene expression microarray
data from the 3-month allograft biopsy as well as eGFR_12 and
CADI_12. A list of candidate genes involved in CAN
progression--that is, genes whose expression in the 3-month
allograft sample correlated with a low eGFR_12 as well as a high
CADI_12--were identified and ranked. SHROOM3 was among the
top-ranked genes on the list (Table 3). Since the interim analysis,
we have collected and analyzed additional samples. At the time of
preparation of the instant application, 3-month allograft gene
expression profiles have been performed on 160 allografts and of
which 12-month eGFR (eGFR 12) was available in 147 subjects and
12-month CADI (CADI_12) was available in 101 subjects (FIG. 1).
When we reexamined the correlation of 3-month allograft SHROOM3
expression in the 147 subjects who had eGFR_12 available, 3-month
allograft SHROOM3 expression correlated inversely with eGFR_12
(r=-0.2192, P<0.01, FIG. 2A). Of the 101 subjects who had
CADI_12, 3-month allograft SHROOM3 expression on gene expression
microarray analysis correlated linearly with CADI_12 (P=0.03, FIG.
2B), which remained significant (P=0.02) when 2 of the allograft
samples were excluded from the analysis--one with BK-virus
nephropathy and another with cortical scarring. No correlation was
identified, however, between 3-month allograft SHROOM3 expression
and simultaneous 3 month CADI (n=135; r=-0.1273, P=0.14, (FIG. 3D).
The relationships of 3-month SHROOM3 expression to CADI_12 and
eGFR_12 were further validated by quantitative real-time polymerase
chain reaction (qRTPCR) analysis in an internal cohort of 32
subjects (r=-0.3873, P=0.02 for eGFR_12 and r=0.3774, P=0.03 for
CADI_12, (FIGS. 3C & 3D). We also found a robust correlation
between microarray and qRTPCR SHROOM3 expression (n=32, r=0.5613;
p=0.0008, (FIGS. 2B and 2C). The relationship between Log-SHROOM3
expression and 12 m-CADI was strongest in DDRs (p<0.01) (FIG.
2D). In multivariate analysis only cold-ischemia time and presence
of acute rejection had significant effect on CADI_12. SHROOM3
expression remained significant in deceased donors (p=0.02). Of the
160 subjects who had 3-month allograft SHROOM3 expression examined
by microarray, 85 had both 3- and 12-month CADI scores available.
To further corroborate that SHROOM3 expression is associated with
progression of CAN we compared SHROOM3 expression between
allografts that had .gtoreq.2 increase in CADI score (n=17;
progressors) to those with less than <2 increments (n=68;
non-progressors) between 3- and 12-month biopsies. SHROOM3
expression was significantly higher in progressors compared to
non-progressors (p=0.04).
Example 2
[0062] Association of SHROOM3 and CAN Progression Exists in
Caucasian-Donor Allografts
[0063] Since a genetic variant of SHROOM3 (SNP variant rs17319721)
is associated with CKD when studied in predominantly Caucasian
cohorts [Kottgen A, 2009], we sought to determine whether the
relationship observed between SHROOM3 and CAN followed a racial
predilection. Of the 147 allografts with available eGFR_12, 109
were from Caucasian donors and their SHROOM3 expression was
inversely correlated with eGFR_12 (r=-0.2712, P=<0.01, FIG. 4A).
Among 101 allografts with available CADI_12, 80 were from Caucasian
donors and SHROOM3 expression in those allografts significantly
correlated with CADI_12 (P=0.02, FIG. 4B). In non-Caucasian
allografts, SHROOM3 expression was not significantly correlated to
either eGFR_12 (n=38) or CADI_12 (n=21) (FIG. 5B, 5C). Among the 85
patients with CADI_3 and CADI_12, 70 received Caucasian-donor
kidneys. In them, we compared Shroom3 expression between
progressors and non-progressors. SHROOM3 expression for the 14
allografts that developed .gtoreq.2 increments in CADI was
significantly higher those with <2 change in CADI (n=56) (FIG.
3C). SHROOM3 expression in the 3-month allograft biopsy was also
significantly different between allografts with varying severity of
CAN based on CADI_12: low (CADI 0-1; n=49), intermediate (CADI 2-5;
n=24), high (CADI>5; n=7) (P=0.03, FIG. 3C).
Example 3
[0064] A Non-Coding SHROOM3 Variant in the Donor is Associated with
Increased SHROOM3 Expression
[0065] The A-allele (minor allele) of a non-coding SHROOM3 SNP at
rs17319721 has been strongly linked to chronic kidney disease by
eGFR-creatinine [Kottgen A, 2009; Boger C, 2011]. We performed
targeted SNP genotyping for the rs17319721 variant in 540 allograft
recipients and 468 donor samples. Allelic prevalence of the risk
allele (A) was 36.66% among recipients and 40.02% among donors. In
both recipients and donors, we observed that the prevalence of the
risk allele was significantly higher in Caucasians compared to
non-Caucasians (P<0.0001) (FIG. 6A). Since a higher SHROOM3
expression correlates with CAN progression and the risk allele of
SHROOM3 is associated with CKD in predominantly Caucasian cohorts,
here we examined whether allograft SHROOM3 expression correlated
with the presence of the risk allele. Of the 160 allografts for
which SHROOM3 expression was available, targeted SNP genotyping of
the rs17319721 variant was performed in 136 cases where either
donor blood samples or pre-perfusion allograft biopsy samples were
available. SHROOM3 expression was significantly higher in
allografts that were homozygous for the risk allele (A/A, n=14)
compared to allografts that were homozygous for G allele (G/G,
n=52, P=0.01, FIG. 6B). SHROOM3 expression of A/G-allografts (n=70)
was not significantly different from A/A or G/G. However, SHROOM3
expression was significantly higher in allografts from donors with
at least one risk allele (A/A or A/G; n=84) compared to donors
without the risk allele (G/G; n=52, P=0.02). When specifically
examined in 103 Caucasian-donor allografts and 33
non-Caucasian-donor allografts the relationship between SHROOM3
expression and the risk allele in the allograft remained constant
but did not attain statistical significance (P=0.05 in
Caucasian-donors FIGS. 7A & 7B respectively). Interestingly,
when we examined the relationship of SHROOM3 expression to
recipient-genotype rather than the donors, there was no significant
correlation between risk allele and expression in 147 recipients
(FIG. 7C).
Example 4
[0066] Risk Allele of SHROOM3 is Associated with Diabetic Mellitus
as the Cause of ESRD and CKD in Caucasians
[0067] Since the risk variant of SHROOM3 has been linked to CKD, we
examined whether the risk allele is associated with a particular
etiology ESRD in our cohort of recipients. As the risk allele of
SHROOM3 was differently distributed between the different
ethnicities (Tables 4a & 4b), we have restricted all subsequent
comparisons between donors and recipients to the same ethnicity. We
observed that the allelic frequency of the risk allele was not
significantly different between Caucasian donors and recipients
(42.87% vs. 43.07% respectively). As only 114 of the 468 donors
were non-Caucasians, the number of subjects in each non-Caucasian
ethnicity (i.e. AAs, Asians, Hispanics and others) was insufficient
to make valid inference about SHROOM3 genotype and its relationship
to ESRD etiology. When we analyzed the allele prevalence among
Caucasian recipients according to their documented ESRD diagnosis
(excluding recipients who had previous transplants, congenital
diseases, or unknown etiology of ESRD), we noted that the risk
allele was most prevalent in Caucasian recipients with diabetes
mellitus as their primary ESRD diagnosis (47.25%; n=126). We found
that Caucasian recipients with diabetes alone without hypertension
had an unadjusted odds ratio of 1.418 (95% CI=1.000-2.11; p=0.04)
while patients with diabetes with hypertension had an odds ratio
1.36 (95% CI=1.031-1.814; p=0.029) of having the risk allele when
both were compared to all Caucasian donors who were not related to
the recipients (FIG. 8A). Furthermore, the allelic prevalence for
the risk allele in patients with diagnosis other than diabetes and
HTN as causes for ESRD was 38.36% (n=159), which was not
significantly different from allelic frequency in unrelated
allograft donors (39.64%). For external validation of the
association of the risk allele with diabetic kidney disease, we
analyzed an independent cohort of subjects. Among the Caucasian
participants of the study (n=3782), 763 were identified as having
CKD. Again allele prevalence was significantly higher in those with
Diabetes and CKD (n=138) compared to those without CKD (n=3019)
and, those without CKD or Diabetes (n=2691) (50.4% vs. 40.4% vs
39.3%, P<0.01) (FIG. 8B). In multivariate analysis within the
Caucasian cohort using age, BMI, hypertension, diabetes, family
history of diabetes or kidney disease as covariates, adjustment for
diabetes negated any effect of the risk allele on CKD as an
outcome. The allele prevalence was also similar in non-diabetics
with and without CKD (38.6% vs 39.26% respectively). Within the CKD
cohort, AAs with CKD (n=721) and, Diabetes with CKD (n=204) had
similar allele distribution as those without CKD (n=2729) (20.59%
vs 18.46% vs 21.78%; P=0.8159) suggesting that the SHROOM3 SNP does
not play a significant role in diabetic/non-diabetic kidney disease
in this group.
Example 5
[0068] Risk-allele of rs17319721 enhances SHROOM3 expression
through TCF4-mediated transcriptional activation rs17319721 is
located within the first intron of SHROOM3. Since the A allele is
associated with a higher expression of SHROOM3, we sought to
understand the effect of the G-to-A substitution on the
transcriptional regulation of SHROOM3. When we examined the
intronic region of SHROOM3 containing rs17319721, we found that the
G-to-A substitution generates a potential consensus binding
sequence for transcription factor 4 (TCF4/TCF7L2), a high mobility
group (HMG) box-containing transcription with the consensus binding
sequence of 5'-(A/T) (A/T)CAAAG-3'. TCF4 is involved in the
Wnt/.beta.-catenin signaling pathway [Wortman B, 2002; Henderson L
J, 2012]. Additionally in our microarray analysis, patients in the
highest quartile of Shroom3 expression also had significantly
upregulated TCF7L2 and Beta-Catenin (P<0.0001). To further
examine whether this intronic region containing the rs17319721 SNP
possesses any function as an enhancer of SHROOM3 transcription, we
generated two SHROOM3 promoter-enhancer luciferase reporter
constructs that consisted of a 3 Kb SHROOM3 promoter region and a
100 bp sequence from the first intron of SHROOM3 containing either
the A-allele or the G-allele of the rs17319721 (FIG. 9. Construct
maps). The SHROOM3 reporter construct with the A-allele had a
higher increase in luciferase-to-renilla reporter activity compared
to the G-allele without TGF.beta. stimulation and with TGF.beta.
stimulation there was an increase in activity. Treatment with
either a TCF4 inhibitor, BC21, or a .beta.-catenin inhibitor
Quercetin, abrogated the difference in reporter activity between
the A- and G-allele reporter constructs (FIG. 10A) which further
confirmed that TCF4-enhancer sequence is responsible for the
difference in the expression of the A- vs G-allele reporter
constructs. To further confirm TCF4 binding to the 100-bp SHROOM3
intronic region we performed EMSA. TCF4 binding to the intronic
sequence containing the A-allele was more than the G-allele, which
was abrogated in samples with excess cold oligos (FIG. 10B)
Example 6
[0069] TGF.beta.1 Enhances SHROOM3 Expression in a
.beta.-Catenin/TCF4-Dependent Manner
[0070] Since SHROOM3 is regulated by TGF.beta.1 in HK-2 cells
[Brennan E P, 2012] and TGF.beta.1 is a key growth factor mediating
renal injury and fibrosis [lan H Y 2012] we further characterized
TGF.beta.1-mediated regulation of SHROOM3. We found that TGF.beta.1
treatment of PRCEC increased SHROOM3 mRNA expression maximally at 5
ng/ml (FIG. 11A) and protein expression at 48 hours (FIG. 11B).
Analysis of the SHROOM3 promoter sequence--up to -10 kb from the
transcriptional start site--using a transcription factor binding
motif prediction program (TRANSFAC) did not reveal any Smad-binding
sequence, suggesting that the TGF.beta.1-induced increase in
SHROOM3 expression is not due to canonical TGF.beta.1/SMAD
signaling. As TGF.beta.1 is known to crosstalk with the
Wnt/.beta.-catenin/TCF4 pathway and TCF4 regulates SHROOM3
expression, we tested whether TGF.beta.1-induced SHROOM3 expression
is dependent on .beta.-catenin/TCF4 interaction. We found that both
BC-21 and quercetin abrogated the TGF.beta.1-induced increase in
the expression of SHROOM3 protein (FIG. 12A) and mRNA (FIG. 12B),
thus confirming that TGF.beta.1-induced SHROOM3 expression is
.beta.-catenin/TCF-4 dependent.
Example 7
[0071] SHROOM3 Facilitates Canonical TGF.beta.1/SMAD3 Signaling and
Profibrotic Gene Expression
[0072] We investigated whether SHROOM3 has any impact on
TGF.beta.1-mediated pro-fibrotic gene program, which is a
well-characterized driver of kidney fibrosis in CKD as well as CAN
[Lan H Y 2012; Campistol J M 2001]. First we compared the
expression of TGF.beta.1-target genes related to tissue fibrosis in
PRCEC with or without SHROOM3 overexpression that were treated with
either TGF.beta.1 or vehicle. Overexpression of SHROOM3 was
confirmed by RTPCR (FIG. 13A) and Western blot (FIG. 13B). The
expression of profibrotic TGF.beta.1-target genes, including COL1A1
and FN1, were increased by TGF.beta.1 treatment alone as well as
SHROOM3 overexpression alone (FIG. 14A). TGF.beta.1-induced
expression of COL1A1 was further increased in cells with SHROOM3
overexpression compared to those without SHROOM3 overexpression
(Vector+TGF). To further characterize how SHROOM3 facilitated
TGF.beta.1 signaling we investigated the phosphorylation of SMAD-3
which indicates activation of canonical TGF.beta.1/SMAD signaling
in PRCEC. Cells with or without SHROOM3 overexpression were treated
with either TGF.beta.1 or vehicle. Phosphorylation of Smad3 in
TGF.beta.1-treated cells was enhanced by SHROOM3 overexpression
compared to vector-transfected cells (FIG. 14B). TGF.beta.1
treatment of SHROOM3-overexpressed PRCEC also developed more
prominent F-actin bundles compared to TGF.beta.1-treated cells
without SHROOM3 overexpression (FIG. 14C-Upper and lower Panels).
Next, we sought to determine whether TGF.beta.1-induced profibrotic
gene program is dependent on SHROOM3. SHROOM3 knockdown in PRCEC
significantly reduced COL1A1 and FN1 transcripts (FIG. 15A).
TGF.beta.1-induced expression of COL1A1 was also significantly
attenuated in SHROOM3 knockdown cells compared to cells transduced
with the empty lentivector. TGF.beta.1-induced expression of FN1,
however, was not affected by SHROOM3 knockdown. Phosphorylation of
SMAD3 in SHROOM3-knockdown cells was significantly reduced at 30
min, but not at 15 min, after TGF.beta.1 stimulation compared to
cells without SHROOM3 knockdown (FIG. 15B). When taken together
these results suggest that SHROOM3 facilitates
TGF.beta.1/SMAD3-induced pro-fibrotic gene expression program.
Further supportive of this was our finding in the microarray cohort
of CTGF, Vimentin, Collagen-IV (downstream of TGF/SMAD3 signaling)
were among genes significantly upregulated in patients in the
highest quartile of SHROOM3 expression.
Example 8
[0073] A-Allele of rs17319721 in the Donor is Associated with
Higher Allograft SHROOM3 Expression at 3 Months.
[0074] Multiple studies have now linked the rs17319721 SHROOM3 SNP
to CKD20-22. Whether the risk allele is associated with altered
SHROOM3 expression in the renal parenchyma, or is related to CAN is
not known. As of Jan. 1, 2013, five hundred eighty nine recipients
have been enrolled in the parent study. We performed targeted
genotyping for this locus on 540 allograft recipients and 517
donors within our cohort. Allelic prevalence of the CKD-associated
A-allele was 36.66% among recipients and 39.94% among donors.
Overall, the prevalence of the A-allele was similar for Caucasian
donors and recipients (42.87% vs. 42.56% respectively). The number
of subjects in each non-Caucasian ethnicity (i.e. AAs, Asians,
Hispanics and others) was not sufficient to make valid inference
about rs17319721 distribution. In both recipients and donors, we
observed that the prevalence of the A-allele is significantly
higher in Caucasians compared to non-Caucasians (P<0.0001).
[0075] Next, we examined whether allograft SHROOM3 expression at
3-months (SHROOM3-3M) correlated with the presence of the A-allele.
Allograft gene expression microarray analysis from 3-month protocol
biopsies was performed on 159 out of the entire 589 enrollees in
this study. These patients represent by chronology the first 159
enrollees who were biopsied 3 months after transplantation. Both
targeted genotyping results for rs17319721 and SHROOM3-transcript
levels from kidney allografts were available from 136 donors and
145 recipients. We observed that SHROOM3-3M was significantly
higher in allografts that were homozygous for the CKD risk allele
(A/A, n=14) compared to allografts that were homozygous for the G
allele (G/G, n=52, P=0.01). SHROOM3-3M was also significantly
higher in allografts from donors with at least one risk allele (A/A
or A/G; n=84) compared to donors without the risk allele (G/G;
n=52, P=0.02). Interestingly, when we examined the relationship of
SHROOM3-3M with respect to the recipient's genotype, rather than
the donor's, there was no significant correlation between the
A-allele and SHROOM3-3M (n=145).
Example 9
[0076] Allograft SHROOM3 Expression at 3-Months and A-Allele of
rs17319721 are Associated with Higher Risk of CAN in Renal
Allograft Recipients.
[0077] Since we observed that the A-allele of rs17319721 is
associated with SHROOM3 transcriptional activation and, that
increased SHROOM3-expression facilitated TGF-.beta.1 signaling in
PRCEC, we examined whether SHROOM3-3M and/or the donor
risk-genotype correlated with indices of allograft dysfunction
(CAN) at 12-months.
[0078] Allograft gene expression microarray analysis from 3-month
protocol biopsies was performed on 159 out of the entire 589
enrollees in this study. At the time of this filing, eGFR-12 was
available in 147 subjects and CADI-12 was available in 101 subjects
from the subgroup. Reasons for not having a 12-month biopsy in this
subgroup included graft loss (n=8), death (n=1), lost-to-follow up
(n=9), contraindication for or inability to obtain a renal
allograft biopsy (n=40).
[0079] SHROOM3-3M correlated inversely with eGFR-12 (r=-0.2192,
P<0.01) and positively with CADI-12 (r=0.2458, P=0.03). This
correlation remained significant (P=0.01) after exclusion of 2
biopsies with diagnosis (BK-virus nephropathy and severe cortical
scarring). The relationship between SHROOM3-3M and CADI-12 was
stronger in deceased-donor allografts (p<0.01). The
relationships of SHROOM3-3M to CADI-12 and eGFR-12 were further
validated by qRTPCR in an internal cohort of 32 subjects (r=-0.39,
P=0.02 for eGFR-12 and r=0.38, P=0.03 for CADI-12). A robust
correlation existed between SHROOM3 expressions from microarray and
qRTPCR (P=0.0008). No correlation existed, however, between
SHROOM3-3M and simultaneous 3-month CADI (n=135). SHROOM3-3M was
predictive of CADI-12 greater than 2 (CADI-12.gtoreq.2) and
inversely related to eGFR-12 in multivariate analysis. Among
covariates included in the analysis, acute rejection before 3
months had significant independent effects on CADI-12, and on
eGFR-12 (P<0.05).
[0080] To corroborate that SHROOM3-3M is associated with
progression of CAN we compared SHROOM3-3M between allografts that
had .gtoreq.2 increase in CADI score (.DELTA.CADI.gtoreq.2, n=17,
known as Progressors) to those with less than <2 change in CADI
score (.DELTA.CADI<2, n=68, known as Non-progressors) between 3-
and 12-month biopsies. To minimize the effect of baseline disease
on subsequent histological progression, we excluded allografts with
CADI-3>2 from this analysis. SHROOM3-3M was significantly higher
in the Progressors compared to the Non-progressors (P=0.04).
[0081] Next we examined the donor risk genotype and its association
with CAN. At the time of analysis, two-hundred and three subjects
of the cohort have had CADI scores reported at 12 months--101 from
the microarray cohort and 102 from the non-microarray cohort. In
this group, the presence of the A-allele in the donor was
associated with a significantly greater risk of a CADI-12.gtoreq.2
in all allografts (OR=1.98, CI=1.10-3.59), indicating a higher risk
of CAN with the risk allele.
Example 10
[0082] SHROOM3 Facilitates Canonical TGF-.beta.1/SMAD3 Signaling
and Profibrotic Gene Expression in a Murine Model In Vivo:
[0083] Methods
[0084] To examine the mechanism of facilitation of fibrosis by
SHROOM3, we developed a murine model of inducible shRNA-mediated
SHROOM3 knockdown. In our model, reverse tetracycline-controlled
transactivator (RTTA)-elements were linked to the universal ROSAm26
promoter for RTTA expression in all cell-types. After in vitro
validation, two SHROOM3-specific shRNA hairpins were linked to
doxycycline-RTTA-responsive elements and positioned 3' to the
Collagen-1 gene. Sample genotyping PCR for Rosa-RTTA element and
shRNA (COLTGM) sequences and southern blot gel electrophoresis are
displayed (FIG. 16A). Doxycycline (DOX) feeding to induce Shroom3
knockdown in genotyped mice was initiated 3-weeks prior to UUO
surgery and continued until the date of sacrifice 10-days later. To
study the development of renal interstitial fibrosis, we performed
unilateral ureteric obstruction surgery (UUO) on 8-10 week old
animals after 3-weeks of DOX-feeding (n=5 in each shRNA clone).
Mice were sacrificed at 10-days post UUO. SHROOM3-shRNA animals of
the same age that were not fed with DOX were used as controls.
Results were analyzed quantitatively by the unpaired t-test.
[0085] Results
[0086] We examined the impact of shRNA-mediated SHROOM3 knockdown
on TGF-.beta.1/SMAD3 signaling and renal fibrosis in the above
DOX-inducible shRNA mouse strain. Doxycycline (DOX) feeding of
these animals confirmed SHROOM3 knockdown (.about.75%) by 3-weeks
(real-time PCR (RT-PCR) and Western blot (WB) from renal cortical
lysates (FIGS. 16B and 16D). DOX-fed animals showed significantly
inhibited phosphorylation of SMAD3 in UUO kidneys by Western blot
(FIG. 16B). COL1A1 production in UUO kidneys was inhibited with
Shroom3 knockdown as shown by RTPCR of kidney lysates and by
immunofluorescence (IF) in tissue sections (FIG. 16C--lower panel
and graph; and 16D). Renal interstitial fibrosis measured by
picrosirius red staining (FIG. 16C--middle panel), was
significantly abrogated in Shroom3 knockdown animals. These results
validate the role of SHROOM3 in canonical TGF-.beta.1
signaling.
[0087] Discussion
[0088] Chronic allograft nephropathy remains a substantial cause
for allograft failure and RRT {Paul L C, 1999} {Chapman J R, 2005}
{Racusen L C, 2010} {USRDS 2012}. Modern immunosuppressive
strategies have had significant impact on short term allograft
outcomes with much less improvement in long-term outcomes
{Hariharan S, 2000} {Meier-Kriesche H U, 2004} {Lamb K E, 2011}.
Further, CAN remains a histological entity with arbitrary stages
and variability between reporting pathologists {Solez K, 2008}
{Akalin E, 2010} {Ying L, 2009} {Halloran P, 2002}. Therefore,
identification of newer markers for potential early diagnosis and
therapy of CAN is imperative. In the GoCAR study, we are examining
the ability of allograft gene-expression profiles from protocol
biopsies at 3-months to predict the development of CAN and TIF at
12 months. We thus identified Shroom3 as a novel candidate gene
whose allograft expression precedes and predicts the derangement of
renal function and the progression of TIF.
[0089] While evolved CAN has shown distinct transcriptional
signatures in prior studies {Flechner S, 2004} {Donauer J, 2003},
issues have been raised regarding their interpretation and
generalizability. Development of biomarkers and/or therapeutic
strategies has been impeded by large gene-panel sizes, small sample
sizes, single time point biopsies, heterogeneity of gene chip assay
used and low fidelity of pre-array amplification techniques {Ying
L, 2009} {Akalin E, 2010}. Studies based on for-cause biopsy
transcriptional profiles are less reliable for developing
predictive panels or therapeutics for CAN due to gene diversity
dependent on pathology at the time of biopsy. To determine a
gene-signature that would predict the development of CAN, Scherer
et al, profiled amplified RNA (Affy HG-U95Av2 chip) from 6-month
protocol biopsies of 17 patients, 12 of whom went on to develop
CAN. They developed a 10-gene cluster that was 88% predictive of
developing chronic rejection at 12-months. The relationship
however, was not significant when single genes were analyzed
{Scherer A, 2003}. We used unamplified RNA from 3-month biopsies
and whole-exon gene chip array (.about.4 probes/exon, .about.40
probes/gene) to correlate differentially expressed genes with eGFR
and CAN at 12 months in our larger cohort of patients. Furthermore,
Shroom3 expression in our study retained its significance when
analyzed alone in the entire cohort with better correlation in the
subset of DDRs and WDKRs. This was validated in our smaller
internal-external cohort of patients by RT-PCR. Importantly, mean
Shroom3 transcript levels were significantly higher in patients
whose CADI-scores progressed (Delta CADI.gtoreq.2) between 3 and 12
months compared to those with relatively stable histological
scores. This implies a role for Shroom3 in the progression of
fibrosis and CAN.
[0090] TIF is a common histological end-point for CAN and CKD.
Consequently, genes linked to fibrogenesis and EC-matrix
production, specifically related to TGF-beta signaling, have
emerged as differentially regulated from transcriptional studies in
patients with CAN and animal models of CKD {Flechner S, 2004}
{Hotchkiss H, 2006} {Mas V, 2007} {Ju W, 2009}. A SNP in Shroom3
has emerged independently linked to incident and prevalent CKD in
Caucasian predominant cohorts {Kottgen A, 2009} {Boger C, 2011}.
However, Shroom3 gene function and its relationship to loss of eGFR
and TIF are hitherto unknown. In PRCEC, our RT-PCR studies suggest
a small but significant overproduction of EC-matrix markers
(Collagen-land Fibronectin) by Shroom3 overexpression and a marked
suppression of these markers with SiRNA mediated Shroom3
inhibition. Further, Shroom3 overexpression appears to facilitate
canonical TGF-beta signaling as evidenced by enhanced Smad-3
phosphorylation in PRCEC. Consistent with this is the amplified
response of profibrotic marker production (SNAIL, MMP-2,
collagen-1) upon TGF-beta treatment in Shroom3-transfected cells
compared to controls. Also in line with these observations are the
suppressed P-Smad3 and Collagen-1 levels we observed with Shroom3
SiRNA in TGF-treated cells. TGF-beta also increased Shroom3
expression in PRCEC. Other groups have made this observation in
HK-2 cells using RNA-sequencing {Brennan E P}. This along with the
salutary effect of increased Shroom3 on TGF-beta signaling may
indicate a positive feedback loop between Shroom3 and TGF.
Together, these suggest that higher Shroom3 expression may have
profibrotic effects in renal epithelial cells. Interestingly, from
our biopsy data, having one or two copies of the risk-allele (A) in
the donor appears to significantly increase Shroom3 transcript
levels in biopsy tissue (.about.1.4 fold). There was no consistent
association between recipient-SNP and Shroom3 expression suggesting
little contribution of allograft Shroom3 expression from
infiltrating recipient cells. In summary, this implies that the
association between the SNP and CKD in prior GWAS studies may be
explained by increased kidney Shroom3 levels and a subsequent
profibrotic response that go along with having the risk allele.
[0091] In univariate analysis the association between Shroom3
expression and eGFR/CADI was significant in WDKRS and DDRs but not
in non-WDKRs or LDRs. Notably, of the DDRs, 51/59 were WDKRS. The
effect--allele prevalence was also significantly higher in whites
compared to non-whites in our analysis. Prior GWAS studies that
identified Shroom3 involved Caucasian predominant cohorts {Kottgen
A, 2009} {Boger C, 2011}. In a study to identify susceptibility
loci for Urinary-albumin creatinine ratio (UACR), the Shroom3 SNP
retained association to eGFR and UACR in whites but did not attain
significance in African-Americans {Ellis J W, 2011}. Our
observation of the lack of association between Shroom3 expression
and eGFR/CADI in non-whites is similar to these published results.
The increased Shroom3 expression with the presence of the A-allele
in the donor, however, was also observed in non-WDKRs. Further the
mean Shroom3 expression by microarray was not significantly
different between WDKRs and non-WDKRS (data not shown). Hence, the
insignificant effect of Shroom3 on eGFR/CADI in non-whites is
unclear. Non-WDKRs in the microarray (n=21) and RT-PCR (n=5) were
fewer than WDKRs. More conjecturally, a polymorphism having greater
impact on allograft outcomes in non-WDKRs that we did not analyze
may be differently distributed within this cohort. In the cohorts
reported on herein, European-Americans with diabetic ESRD and CKD
respectively had the highest prevalence of the risk allele of
rs17319721. However, the Family Investigation of Nephropathy and
Diabetes (FIND) study did not report linkage between loci on
chromosome 4 and diabetic nephropathy in Caucasians {Igo R P,
2011}. The inconsistency between these results may stem from our
comparison group which included only kidney donors who were not
related.
[0092] All patients in the 3-month microarray did not have biopsies
for outcome assessment at 12-months (17 allograft losses, 42 lost
follow-up or refused 12-month biopsy). The sample size of 101
patients is still robust in comparison to prior transcriptional
studies in allograft recipients {Flechner S, 2004} {Donauer J,
2003} {Sarwal M, 2003} {Reeve J, 2009}. Only 36 patients had
sufficient quality RNA for RT-PCR validation after
microarray--though the relationships were significant within this
sample. While the facilitation of TGF-beta signaling by Shroom3
excess was observed in vitro, the mechanism of this interaction is
uncertain. The C-terminal ASD-2 domain of Shroom3 has been shown to
be essential for Rho-Kinases 1&2 (ROCKs) recruitment and
function in the invaginating neural tube. Mutation of this domain
leads to loss of ROCK-function {Nishimura, 2008}. In chondrocytes,
ROCKs facilitated and ROCK-inhibitor (Y27632) inhibited
Smad3-phosphorylation with TGF-beta treatment {Xu T, 2012}.
Further, in animal models of unilateral ureteral obstruction,
ROCK-inhibitors retarded renal interstitial fibrosis and
GFR-decline {Satoh S, 2002} {Nagatoya K, 2002} {Takeda Y, 2010}.
Though we did not test this, the mechanism of interaction with
TGF-beta may in part be through Shroom3-mediated ROCK facilitation.
Finally, the rs17319721 locus on the Shroom3 gene is intronic,
between exons 1 and 2. The reason for the apparent regulatory
effects of this region is less clear from our observations.
Recently, the results from the Encyclopedia of DNA elements
(ENCODE) consortium have been sequentially published describing the
regulatory functions attached to intronic loci within the human
genome {Dunham I, 2012} {Neph S, 2012} {Gerstein M, 2012}. Studies
using histone methylation mapping suggest that the Shroom3 SNP is
located in an area predicted to have enhancer function {Dr Katalina
Susztak, ASN 2012}. This is consistent with our findings.
[0093] In summary, Shroom3 is a novel candidate gene whose
expression in the renal allograft precedes and predicts the
derangement of renal function and TIF in CAN. Higher allograft
Shroom3 levels appear to predict histological progression of CAN.
We also show that these relationships are best in recipients of
white-donor kidneys and deceased-donor kidneys. Our findings
confirm for the first time that the previously described
CKD-associated Shroom3 locus (rs17319721) {Kottgen A, 2009} {Boger
C, 2011} mediates its effect through increased Shroom3 expression.
Finally our in vitro studies suggest a salutary role for Shroom3 in
canonical TGF-beta signaling and type I collagen production promise
as a therapeutic target in both CAN and CKD to reduce the
progression of TIF and retard ESRD.
[0094] The present invention also includes a kit for use in
identifying patients suffering from kidney diseases and for
predicting the progression of kidney fibrosis and TIF in renal
allograft recipients. The kit consists of reagents for RT-PCT
analysis of Shroom3 expression and a microassay for detecting the
rs 17319721 SNP risk allele, a positive control for the RT-PCR
assays, buffers and instructions for use. The kit is used by
obtaining a renal biopsy from a patient who received a kidney
transplant from a donor, determining Shroom3 expression and
comparing a level of Shroom3 expression in biopsy specimen with the
level of Shroom expression in the positive control in the kit, all
by RT-PCR. The microarray is used to identify kidney donors who are
homozygous for the Shroom3 A allele.
TABLE-US-00001 TABLE 1 Demographics of GOCAR enrollees Mean .+-. SD
Demographics N (%) [range] Recipient Age: All Recipients 589 50.17
yrs [18-83] Recipient Gender (Percent females) 185 (31.41)
Recipient Race: White (W) 375 (63.37) African-American (AA) 123
(20.88) Asian (A) 34 (5.77) Hispanic (H) 34 (5.77) Other (O) 16
(2.71) Recipient ESRD diagnosis- All recipients: Diabetes only 115
(19.42) Hypertension only 97 (15.28) Diabetes & Hypertension 90
(15.28) Polycystic disease 53 (8.99) Glomerular disease (including
107 (18.17) FSGS/IgA) Unknown 16 (2.72) Prior transplants 13 (2.38)
Others 98 (16.64) Donor Age 589 42.01 yrs [0-76] Donor Gender
(Percent females) 278 (47.20) Donor Race: White (W) 451 (76.57)
African-American (AA) 57 (9.68) Asian (A) 17 (2.89) Hispanic (H) 45
(7.64) Other (O) 19 (3.23) Donor type: Deceased-Donors (DD) 329
(55.86) Living-Related Donors (LRD) 147 (24.96) Living-Unrelated
Donors (LURD) 113 (19.19) Donor SNP analysis 468 Recipient SNP
analysis 540
TABLE-US-00002 TABLE 2 Demographics of patients in the microarray
studies (CADI & eGFR analysis) Mean .+-. SD Demographics N (%)
[range] Recipient Age: 160 (100) 48.84 .+-. 13.27 [19-73] Recipient
Gender (Percent 47 (29.35) females) Recipient Race: White (W) 92
(57.5) African-American (AA) 37 (23.1) Asian (A) 10 (6.25) Hispanic
(H) 14 (8.75) Other (O) 7 (4.38) Donor Age 160 41.13 .+-. 16.80 yrs
[3-76] Donor Gender 77 (48.13) (Percent females) Donor Race: White
(W) 121 (75.63) African-American (AA) 17 (10.63) Asian (A) 7 (4.38)
Hispanic (H) 12 (7.5) Other (O) 3 (1.88) Donor type:
Deceased-Donors (DD) 95 (59.38) Living-Related Donors (LRD) 35
(21.88) Living-Unrelated Donors 30 (18.75) (LURD) CADI analysis
(3-months) 135 1.18 .+-. 1.76 [0-9] CADI analysis (12-months) 101
2.09 .+-. 2.49 [0-10] eGFR analysis (6-months) 139 57.40 .+-. 17.4
ml/min [15.08-116.37] eGFR analysis (12-months) 147 58.12 .+-.
19.45 ml/min [10.66-109.24]
TABLE-US-00003 TABLE 3a SNP distribution in genotyped Donors and
Recipients in the GoCAR cohort (Donors = 468, Recipients = 540)
Whites Non-Whites Genotypes Donor Recipient Donor Recipient A/A 57
(16.1) 59 (16.7) 13 (11.4) 10 (5.4) A/G 190 (53.7) 187 (52.8) 44
(38.6) 65 (34.9) G/G 107 (30.2) 108 (30.50) 57 (50) 109 (58.7)
A-allele (%) 42.86 43.07 41.67 30.56 Total 354 354 114 186
TABLE-US-00004 TABLE 3B Demographics & Race-wise distribution
of non-white donors (114) and non- white recipients (186)
Afro-American Hispanic Asian Other* Genotypes Donor Recipient Donor
Recipient Donor Recipient Donor Recipient A/A 1 (1.8) 4 (3.6) 9
(21.9) 6 (19.4) 1 (7.1) 0 (0) 2 (33.3) 2 (11.1) A/G 23 (43.4) 43
(39.1) 17 (41.5) 12 (38.7) 3 (21.4) 3 (11.1) 1 (16.6) 7 (38.9) G/G
29 (54.8) 63 (57.3) 15 (36.6) 13 (41.9) 10 (71.4) 24 (88.9) 3 (50)
9 (50) A-allele (%) 23.58 23.18 42.68 38.70 17.85 11.11 41.67 30.56
Total 53 110 41 31 14 27 6 18
TABLE-US-00005 TABLE 4 QPCr primer sequences Forward primer Reverse
primer Gene name (5' .fwdarw. 3') (5' .fwdarw. 3') HGAPDH
TGTTGCCATCAATGA CTCCACGACGTACTC CCCCTT AGCG (SEQ ID NO: 1) (SEQ ID
NO: 2) Shroom3 CCCTCTCGGGGCGTC GCCCAGCACTACTCG TAGCC CTCC (SEQ ID
NO: 3) (SEQ ID NO: 4) Collagen-1 GATGGTGAAGATGGT GCCCAAGTCCAACTC
CCCAC CTTTT (SEQ ID NO: 5) (SEQ ID NO: 6) Fibronecctin-1
TCCAGGAGTTCACTG CTGCAAGCCTTCAAT TGCC AGTCA (SEQ ID NO: 7) (SEQ ID
NO: 8) SNAIL ACCACTATGCCGCGC GGTCGTAGGGCTGCT TCTT GGAA (SEQ ID NO:
9) (SEQ ID NO: 10) Matrix ACCCAGATGTGGCCA GAGCAAAAGGCATCA Metallo-
ACTAC TCCACT proteinase-2 (SEQ ID NO: 11) (SEQ ID NO: 12) Vimentin
TTGACCTTGAACGCA GCTGTTCCTGAATCT AAGTG GAGCC (SEQ ID NO: 13) (SEQ ID
NO: 14) E-Cadherin CAGCACGTACACAGC ACCTGAGGCTTTGGA CCTAA TTCCT (SEQ
ID NO: 15) (SEQ ID NO: 16) Slug GATGCATATTCGGAC CCTCATGTTTGTGCA
CCACAC GGAGAG (SEQ ID NO: 17) (SEQ ID NO: 18) FSP-1 GCCCTGGATGTGATG
TCGTTGTCCCTGTTG GTGT CTGTC (SEQ ID NO: 19) (SEQ ID NO: 20) SMA
CAACCGGGAGAAAAT TAGATGGGGACATTG GACTC TGGGT (SEQ ID NO: 21) (SEQ ID
NO: 22)
TABLE-US-00006 TABLE 5 Primer sequences designed for RT-PCR and
generating Luciferase-Reporter plasmid constructs Forward primer
Reverse primer Primers (5' .fwdarw. 3') (5' .fwdarw. 3') HGAPDH
TGTTGCCATCAATGA CTCCACGACGTACTC CCCCTT AGCG (SEQ ID NO: 23) (SEQ ID
NO: 24) SHROOM3 CCCTCTCGGGGCGTC GCCCAGCACTACTCG TAGCC CTCCC (SEQ ID
NO: 25) (SEQ ID NO: 26) Constructs TTATAGGTACCTTGA TTAAGCTTCCATGCC
Wild-type GACAATAGAGTTGCC AAACACATGATCCCT (promoter (SEQ ID NO: 27)
C only) (SEQ ID NO: 28) A-allele TTTGGTACCGAGTAG TTAAGCTTCCATGCC
Construct CAGGGCAAAAACAAA AAACACATGATCCCT AGCCCTTGAGACAAT C
AGAGTTGCC (SEQ ID NO: 30) (SEQ ID NO: 29) G-Allele TTTGGTACCGAGTAG
TTAAGCTTCCATGCC Construct CAGGGCAAAAACAAA AAACACATGATCCCT
GGCCCTTGAGACAAT C AGAGTTGCC (SEQ ID NO: 32) (SEQ ID NO: 31) Legend:
Kpnl was introduced in all forward primer and Hind III in the
Reverse ones.
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[0149] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0150] It is further to be understood that all values are
approximate, and are provided for description. Patents, patent
applications, publications, product descriptions, and protocols are
cited throughout this application, the disclosures of which are
incorporated herein by reference in their entireties for all
purposes.
Sequence CWU 1
1
32121DNAArtificial Sequencesynthetically generated oligonuelcotides
1tgttgccatc aatgacccct t 21219DNAArtificial Sequencesynthetically
generated oligonuelcotides 2ctccacgacg tactcagcg 19320DNAArtificial
Sequencesynthetically generated oligonuelcotides 3ccctctcggg
gcgtctagcc 20419DNAArtificial Sequencesynthetically generated
oligonuelcotides 4gcccagcact actcgctcc 19520DNAArtificial
Sequencesynthetically generated oligonuelcotides 5gatggtgaag
atggtcccac 20620DNAArtificial Sequencesynthetically generated
oligonuelcotides 6gcccaagtcc aactcctttt 20719DNAArtificial
Sequencesynthetically generated oligonuelcotides 7tccaggagtt
cactgtgcc 19820DNAArtificial Sequencesynthetically generated
oligonuelcotides 8ctgcaagcct tcaatagtca 20919DNAArtificial
Sequencesynthetically generated oligonuelcotides 9accactatgc
cgcgctctt 191019DNAArtificial Sequencesynthetically generated
oligonuelcotides 10ggtcgtaggg ctgctggaa 191120DNAArtificial
Sequencesynthetically generated oligonuelcotides 11acccagatgt
ggccaactac 201221DNAArtificial Sequencesynthetically generated
oligonuelcotides 12gagcaaaagg catcatccac t 211320DNAArtificial
Sequencesynthetically generated oligonuelcotides 13ttgaccttga
acgcaaagtg 201420DNAArtificial Sequencesynthetically generated
oligonuelcotides 14gctgttcctg aatctgagcc 201520DNAArtificial
Sequencesynthetically generated oligonuelcotides 15cagcacgtac
acagccctaa 201620DNAArtificial Sequencesynthetically generated
oligonuelcotides 16acctgaggct ttggattcct 201721DNAArtificial
Sequencesynthetically generated oligonuelcotides 17gatgcatatt
cggacccaca c 211821DNAArtificial Sequencesynthetically generated
oligonuelcotides 18cctcatgttt gtgcaggaga g 211919DNAArtificial
Sequencesynthetically generated oligonuelcotides 19gccctggatg
tgatggtgt 192020DNAArtificial Sequencesynthetically generated
oligonuelcotides 20tcgttgtccc tgttgctgtc 202120DNAArtificial
Sequencesynthetically generated oligonuelcotides 21caaccgggag
aaaatgactc 202220DNAArtificial Sequencesynthetically generated
oligonuelcotides 22tagatgggga cattgtgggt 202321DNAArtificial
Sequencesynthetic primer 23tgttgccatc aatgacccct t
212419DNAArtificial Sequencesynthetic primer 24ctccacgacg tactcagcg
192520DNAArtificial Sequencesynthetic primer 25ccctctcggg
gcgtctagcc 202620DNAArtificial Sequencesynthetic primer
26gcccagcact actcgctccc 202730DNAArtificial Sequencesynthetic
primer 27ttataggtac cttgagacaa tagagttgcc 302831DNAArtificial
Sequencesynthetic primer 28ttaagcttcc atgccaaaca catgatccct c
312954DNAArtificial Sequencesynthetic primer 29tttggtaccg
agtagcaggg caaaaacaaa agcccttgag acaatagagt tgcc
543031DNAArtificial Sequencesynthetic primer 30ttaagcttcc
atgccaaaca catgatccct c 313154DNAArtificial Sequencesynthetic
primer 31tttggtaccg agtagcaggg caaaaacaaa ggcccttgag acaatagagt
tgcc 543231DNAArtificial Sequencesynthetic primer 32ttaagcttcc
atgccaaaca catgatccct c 31
* * * * *
References