U.S. patent application number 14/266935 was filed with the patent office on 2015-02-12 for global dna hypomethylation and biomarkers for clinical indications in cancer.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY. The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to Rafael Guerrero-Preston, David Sidransky.
Application Number | 20150045241 14/266935 |
Document ID | / |
Family ID | 47627305 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150045241 |
Kind Code |
A1 |
Guerrero-Preston; Rafael ;
et al. |
February 12, 2015 |
GLOBAL DNA HYPOMETHYLATION AND BIOMARKERS FOR CLINICAL INDICATIONS
IN CANCER
Abstract
The present invention provides methods of determination of a
global DNA methylation index (GDMI) in a sample from a subject,
using a variety of methods which can detect global, genome-wide,
and gene-specific DNA methylation to create methylation portraits
that can be used for early detection, diagnosis, and clinical
management in the personalized medicine space. Further, the
invention provides methods of diagnosis of cancer, including
gastric cancer and hepatocellular cancer in a subject, by comparing
the GDMI in a sample obtained from a subject to the methylation
index of standard controls. These methods allow diagnosis of
gastric carcinoma and liver cancer in patients who may be
asymptomatic or have inconclusive pathology, and allowing earlier
treatment of the subject.
Inventors: |
Guerrero-Preston; Rafael;
(Baltimore, MD) ; Sidransky; David; (Baltimore,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
Baltimore |
MD |
US |
|
|
Assignee: |
THE JOHNS HOPKINS
UNIVERSITY
Baltimore
MD
|
Family ID: |
47627305 |
Appl. No.: |
14/266935 |
Filed: |
May 1, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13408338 |
Feb 29, 2012 |
8728732 |
|
|
14266935 |
|
|
|
|
61448020 |
Mar 1, 2011 |
|
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Current U.S.
Class: |
506/9 ;
435/6.11 |
Current CPC
Class: |
G01N 2800/50 20130101;
C12Q 1/6886 20130101; C12Q 2600/154 20130101; C12Q 1/6827
20130101 |
Class at
Publication: |
506/9 ;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with U.S. government support under
one or more of the following grant nos. U01CA84986, P50DE019032,
RC2 DE20957, 5U01CA13-8, 1RC2 DE20957, 5T32CA009529-20, 2P30AG
15294, CA 146799, DK087454, CA 133012, and 5S21 MD008130-02 awarded
by the National Institutes of Health. The U.S. government has
certain rights in the invention.
Claims
1. A method for obtaining a global DNA methylation index (GDMI) in
a sample comprising: a) obtaining a biological sample comprising
DNA from a subject; b) determining the amount of 2'-deoxycytidine
(2dc) and 5-methyl-2'-deoxycytidine (5mdc) in the DNA sample; c)
generating a GDMI for the sample by comparing the amount of 5mdc
relative to the amount of 5mdc+2dc in the sample and generating a
ratio.
2. The method of claim 1, wherein the method of for making the
determination of b) is selected from the group consisting of
quantitative methylation specific PCR (qMSP), oligonucleotide
methylation tiling arrays, methylation BeadChip assays, ELISA, and
the use of HPLC/MS.
3. A method for identifying whether a subject has an increased risk
of hepatocellular carcinoma (HCC) comprising: a) obtaining a
biological sample of hepatocytes comprising DNA from a subject; b)
determining the amount of 2'-deoxycytidine (2dc) and
5-methyl-2'-deoxycytidine (5mdc) in the DNA sample; c) generating a
global DNA methylation index (GDMI) for the sample by comparing the
amount of 5mdc relative to the amount of 5mdc+2dc in the sample and
generating a ratio; and d) comparing the GDMI of the sample to the
GDMI of a control, wherein when the GDMI of the sample is less than
the GDMI of the control, then the subject has an increased risk of
HCC; and e) identifying the appropriate treatment regimen for the
subject.
4. The method of claim 3, wherein the method of for making the
determination of b) is selected from the group consisting of
quantitative methylation specific PCR (qMSP), oligonucleotide
methylation tiling arrays, methylation BeadChip assays, ELISA, and
the use of HPLC/MS.
5. The method of claim 3, wherein the subject is infected with HBV
or HCV.
6.-8. (canceled)
9. A method for identifying whether a subject has an increased risk
of gastric carcinoma (GC) comprising: a) obtaining a biological
sample of the gastric mucosa comprising DNA from a subject; b)
determining the amount of 2'-deoxycytidine (2dc) and
5-methyl-2'-deoxycytidine (5mdc) in the DNA sample; c) generating a
global DNA methylation index (GDMI) for the sample by comparing the
amount of 5mdc relative to the amount of Smdc+2dc in the sample and
generating a ratio; and d) comparing the GDMI of the sample to the
GDMI of a control, wherein when the GDMI of the sample is less than
the GDMI of the control, then the subject has an increased risk of
GC; and e) identifying the appropriate treatment regimen for the
subject.
10. The method of claim 9, wherein the method of for making the
determination of b) is selected from the group consisting of
quantitative methylation specific PCR (qMSP), oligonucleotide
methylation tiling arrays, methylation BeadChip assays, ELISA, and
the use of HPLC/MS.
11. A method for differentiating whether a subject has a high risk
of developing GC or a low risk of developing GC in a subject who is
diagnosed with deep gastric inflammation comprising: a) obtaining a
biological sample of gastric tissue comprising DNA from the
subject; b) determining the amount of 2'-deoxycytidine (2dc) and
5-methyl-2'-deoxycytidine (5mdc) in the DNA sample; c) generating a
GDMI for the sample by comparing the amount of 5mdc relative to the
amount of Smdc+2dc in the sample and generating a ratio; and d)
comparing the GDMI of the sample to the GDMI of a control, wherein
when the GDMI of the sample is less than the GDMI of the control,
then the subject has a high risk of GC; and e) identifying the
appropriate treatment regimen for the subject.
12. The method of claim 11, wherein the method of for making the
determination of b) is selected from the group consisting of
quantitative methylation specific PCR (qMSP), oligonucleotide
methylation tiling arrays, methylation BeadChip assays, ELISA, and
the use of HPLC/MS.
13. The method of claim 11, wherein the subject is ambulatory.
14. The method of claim 11, wherein the sample is obtained or
assessed with an endoscope.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional application of U.S. patent
application Ser. No. 13/408,338, filed Feb. 29, 2012, now U.S. Pat.
No. 8,728,732, issued May 20, 2014, which claims the benefit of
U.S. Provisional Application No. 61/448,020, filed Mar. 1, 2011,
the content of each of the aforementioned applications is herein
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Epigenomics refers to the inheritance of information based
on gene expression levels that do not entail changes in DNA
sequence, as opposed to genetics which refers to information
transmitted on the basis of gene sequence. The best understood
epigenomic marks include DNA methylation, histone modifications,
and micro-RNA (miRNA). Epigenomics has been called the science of
change. It is a biological endpoint for endogenous and exogenous
factors that determine health and disease.
[0004] DNA methylation is one of the most common alterations in
human neoplasia, including breast cancer. DNA methylation refers to
the addition of a methyl group to the cytosine ring of those
cytosines that precede guanosine (CpG dinucleosides) to form methyl
cytosine. Detection of changes in DNA methylation may offer an
alternative to screening and may offer data for long-term
management of women treated for breast cancer.
[0005] Hepatocellular carcinoma (HCC) is the most common primary
malignancy of the liver in adults, the fifth most common solid
tumor, and the third most common cause of cancer death worldwide.
HCC incidence and death rates are steadily rising in the United
States and demonstrate the highest average annual percent increase
of the top 15 cancers by incidence. HCC patients, and people at
risk of developing HCC, have profound unmet medical and public
health needs. Advances in HCC treatment such as, liver
transplantation, surgical resection, and loco regional therapies
have only impacted a fraction of the population at risk. More than
70% of HCC patients present with advanced disease and will not
benefit from these treatment modalities, or from the sole
chemotherapeutic agent approved for advanced HCC patients.
[0006] Most HCC patients cannot benefit from current treatment
modalities because they are diagnosed with advance disease. The
obesity epidemic and the increase in HCV related cirrhosis will
eventually lead to an increase in the incidence of HCC. The
projected increase in HCC incidence creates an urgent need for
translational research that leads to novel screening and clinical
management for HCC. HCC mortality, however, can be decreased by
effective early detection strategies followed by curative treatment
such as resection, liver transplantation, or liver ablation. The
smaller the HCC tumor is at diagnosis, the higher the likelihood of
therapeutic success. Consequently, the goal of screening programs
for at risk populations is to detect and treat HCC at an early
stage and, specifically, when tumors can be detected before they
have grown larger than 2 cm in diameter.
[0007] According to American Association for the Study of Liver
Diseases (AASLD) guidelines, HCC screening should be done with
ultrasound every 6-12 months. HCC diagnosis requires a specific
algorithm with dynamic imaging techniques for the characterization
of liver nodules detected during surveillance of patients with
cirrhosis with ultrasound (US). A combination of dynamic contrast
imaging techniques, contrast-enhanced ultrasound (CE-US), computed
tomography (CT), and gadolinium magnetic resonance imaging (MRI),
are considered the standard of care for the radiological diagnosis
of HCC in cirrhotic patients. However, the accuracy of radiological
diagnosis depends largely on the degree of arterial
hypervascularization, which increases with tumor size, and also by
cell grading of the nodule. HCC diagnostic accuracy could be
improved if a molecular biomarker was identified that could
distinguish HCC from non-HCC cells in the nodule.
[0008] Similar to HCC, gastric cancer (GC) is the fourth most
common cancer in both sexes and the second cause of cancer-related
death around the world. The prognosis of GC is closely related to
the stage of disease at the time of diagnosis. Early GC is defined
as cancer confined to the mucosa or submucosa regardless of the
presence of lymph node metastasis. Apart from conventional,
magnifying narrow-band imaging (NBI), endoscopy has been recently
introduced for the diagnosis of early GC. However, missed diagnoses
of GC on endoscopy are still common, with false-negative rates
ranging from 5 to 19%. Whereas the five year survival rate for
early GC is greater than 90%, prognosis for advanced GC is still
poor. A contributing factor to this poor prognostication rate is
the difficulty in distinguishing early GC from benign peptic ulcer
or gastritis in the ambulatory setting, as most of the patients
with early GC do not have specific symptoms. Due to the above
mentioned reasons, less than 20% of GCs are diagnosed at an early
stage in several countries.
[0009] The integration of epigenomics and DNA methylation to
clinical and population based studies is still lacking. As such,
there still exists a need for better clinical methods for
determining biomarkers useful in detection and diagnosis of hepatic
and gastric cancers.
SUMMARY OF THE INVENTION
[0010] In an embodiment, the present invention provides a method
for obtaining a global DNA methylation index (GDMI) in a sample
comprising a) obtaining a biological sample comprising DNA from a
subject, b) determining the amount of 2'-deoxycytidine (2dc) and
5-methyl-2'-deoxycytidine (5mdc) in the DNA sample, c) generating a
GDMI for the sample by comparing the amount of 5mdc relative to the
amount of Smdc+2dc in the sample and generating a ratio.
[0011] In another embodiment, the present invention provides a
method for identifying whether a subject has an increased risk of
HCC comprising a) obtaining a biological sample of hepatocytes
comprising DNA from a subject, b) determining the amount of
2'-deoxycytidine (2dc) and 5-methyl-2'-deoxycytidine (5mdc) in the
DNA sample, c) generating a GDMI for the sample by comparing the
amount of 5mdc relative to the amount of Smdc+2dc in the sample and
generating a ratio and d) comparing the GDMI of the sample to the
GDMI of a control, wherein when the GDMI of the sample is less than
the GDMI of the control, then the subject has an increased risk of
HCC.
[0012] In a further embodiment, the present invention provides a
method of diagnosis of HCC in a subject suspected of having HCC
comprising a) obtaining a biological sample of hepatocytes
comprising DNA from the subject, b) detecting the amount of
promoter methylation on at least one or more DNA target sites
selected from the group consisting of RASSF1A, SSBP2, and B4GALT1,
and c) comparing the amount of promoter methylation on at least one
or more DNA target sites in the sample of the subject to the amount
of promoter methylation in a control sample, wherein when the
amount of promoter methylation on at least one or more DNA target
sites is less than the amount of promoter methylation in the
control sample, the patient is diagnosed as having HCC.
[0013] In yet another embodiment, the present invention provides a
method for identifying whether a subject has an increased risk of
GC comprising a) obtaining a biological sample of the gastric
mucosa comprising DNA from a subject, b) determining the amount of
2'-deoxycytidine (2dc) and 5-methyl-2'-deoxycytidine (5mdc) in the
DNA sample, c) generating a GDMI for the sample by comparing the
amount of 5mdc relative to the amount of 5mdc+2dc in the sample and
generating a ratio, and d) comparing the GDMI of the sample to the
GDMI of a control, wherein when the GDMI of the sample is less than
the GDMI of the control, then the subject has an increased risk of
GC.
[0014] In still a further embodiment, the present invention
provides a method for differentiating whether a subject has a high
risk of developing GC or a low risk of developing GC in a subject
who is diagnosed with deep gastric inflammation comprising a)
obtaining a biological sample of gastric tissue comprising DNA from
a subject, b) determining the amount of 2'-deoxycytidine (2dc) and
5-methyl-2'-deoxycytidine (5mdc) in the DNA sample, c) generating a
GDMI for the sample by comparing the amount of 5mdc relative to the
amount of 5mdc+2dc in the sample and generating a ratio and d)
comparing the GDMI of the sample to the GDMI of a control, wherein
when the GDMI of the sample is less than the GDMI of the control,
then the subject has a high risk of GC.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a histogram depicting the GDMI in cases and
controls study groups.
[0016] FIG. 2 is a histogram depicting GDMI in gastritis patients
with superficial inflammation and deep inflammation.
[0017] FIG. 3 is a histogram showing GDMI in gastritis patients
with deep inflammation who are positive and negative for intestinal
metaplasia, a premalignant lesion which may lead to GC.
[0018] FIG. 4 is a table listing the QMSP Primers and probes used
in the present invention.
[0019] FIG. 5A is a table showing hepatocellular carcinoma risk
factors for participants 1-30 in the HCC studies. FIG. 5B is a
table showing hepatoccllular carcinoma risk factors for
participants 31-43 in the HCC studies.
[0020] FIG. 6 comprises scatterplots and histograms for a
representative set of one tumor sample and two normal samples
hybridized to oligonucelotide methylation tiling arrays. The
methylation score is on the Y axis of the scatterplots and the
number of methylated probes is on the X axis. The number of
methylated probes is on the Y axis of the histograms and the
methylation score is on the X axis.
[0021] FIG. 7 shows quantitative methylation specific PCR (QMSP)
results of Hepatocellular carcinoma samples and adjacent normal
liver samples that were bisulfite treated to examine the promoter
methylation status of RASF1A, B4GALT11 and SSBP2. Scatter plots of
QMSP analysis of candidate gene promoters. Eighteen adjacent normal
liver tissue samples and 27 hepatocellular carcinoma samples were
tested for methylation for each of the three genes by quantitative
MSP. The relative level of methylated DNA for each gene in each
sample was determined as a ratio of MSP for the amplified gene to
ACTB and then multiplied by 100 for easier tabulation (average
value of duplicates of gene of interest/average value of duplicates
of ACTB).times.100. The samples were categorized as unmethylated or
methylated based on detection of methylation above a threshold set
for each gene. This threshold was determined by analyzing the
levels and distribution of methylation, if any, in normal,
age-matched tissues.
[0022] FIG. 8A depicts ROC curves for a panel of the three genes
RASSF1A, B4GALT1 and SSBP2 by themselves. FIG. 8B depicts ROC
curves for the same panel of the three genes RASSF1A, B4GALT1 and
SSBP2, after adjusting a logistic regression model with HCC risk
factors: age, gender, ethnicity and etiology.
DETAILED DESCRIPTION OF THE INVENTION
[0023] One of the aims of the present invention was to develop a
Phase I biomarker using the GDMI, and determine whether it could
distinguish HCC and GC cases from controls in two ambulatory
clinics that monitor a great number of high risk patients diagnosed
with premalignant or early hepatic and gastric cancer lesions.
[0024] In an embodiment, the present invention provides a method
for obtaining a GDMI in a sample comprising a) obtaining a
biological sample comprising DNA from a subject, b) determining the
amount of 2'-deoxycytidine (2dc) and 5-methyl-2'-deoxycytidine
(5mdc) in the DNA sample, c) generating a GDMI for the sample by
comparing the amount of 5mdc relative to the amount of 5mdc+2dc in
the sample and generating a ratio.
[0025] In accordance with another embodiment of the present
invention, it will be understood that the term "biological sample"
or "biological fluid" includes, but is not limited to, any quantity
of a substance from a living or formerly living patient or mammal.
Such substances include, but are not limited to, blood, serum,
plasma, urine, cells, organs, tissues, bone, bone marrow, lymph,
lymph nodes, synovial tissue, chondrocytes, synovial macrophages,
endothelial cells, and skin.
[0026] It will be understood by those of ordinary skill, that there
are a number of ways to detect DNA methylation, and these are known
in the art. Examples of preferred methods of detection of
methylation of DNA in a sample include the use of QMSP,
oligonucleotide methylation tiling arrays, paramagnetic beads
linked to MBD2, i.e., BeadChip assays and HPLC/MS methods. Other
methods include methylation-specific multiplex ligation-dependent
probe amplification (MS-MPLA), bisulfate sequencing, and assays
using antibodies to DNA methylation, i.e., ELISA assays. The
methylation state or GDMI information gathered from these methods
can be generated using any type of microprocessor or computing
device.
[0027] As used herein, the term "methylation state" means the
detection of one or more methyl groups on a cytidine in a target
site of the DNA in the sample.
[0028] By "nucleic acid" as used herein includes "polynucleotide,"
"oligonucleotide." and "nucleic acid molecule," and generally means
a polymer of DNA or RNA, which can be single-stranded or
double-stranded, synthesized or obtained (e.g., isolated and/or
purified) from natural sources, which can contain natural,
non-natural or altered nucleotides, and which can contain a
natural, non-natural or altered internucleotide linkage, such as a
phosphoroamidate linkage or a phosphorothioate linkage, instead of
the phosphodiester found between the nucleotides of an unmodified
oligonucleotide. It is generally preferred that the nucleic acid
does not comprise any insertions, deletions, inversions, and/or
substitutions. However, it may be suitable in some instances, as
discussed herein, for the nucleic acid to comprise one or more
insertions, deletions, inversions, and/or substitutions.
[0029] It will be understood that the methods of the present
invention which determine the methylation state of a sample of DNA
(GDMI) are useful in preclinical research activities as well as in
clinical research in various diseases or disorders, including, for
example, cancer.
[0030] In accordance with an embodiment, the present invention
provides a method for identifying whether a subject has an
increased risk of HCC comprising a) obtaining a biological sample
of hepatocytes comprising DNA from a subject, b) determining the
amount of 2'-deoxycytidine (2dc) and 5-methyl-2'-deoxycytidine
(5mdc) in the DNA sample, c) generating a GDMI for the sample by
comparing the amount of 5mdc relative to the amount of 5mdc+2dc in
the sample and generating a ratio and d) comparing the GDMI of the
sample to the GDMI of a control, wherein when the GDMI of the
sample is less than the GDMI of the control, then the subject has
an increased risk of HCC.
[0031] Conversely, if the GDMI of the sample is the same or greater
than the GDMI of the control, then the subject does not have an
increased risk of developing HCC.
[0032] In accordance with an embodiment, the present invention
provides a method of diagnosis of HCC in a subject suspected of
having HCC comprising a) obtaining a biological sample of
hepatocytes comprising DNA from the subject, b) detecting the
amount of promoter methylation on at least one or more DNA target
sites selected from the group consisting of RASSF1A, SSBP2, and
B4GALT1, and c) comparing the amount of promoter methylation on at
least one or more DNA target sites in the sample of the subject to
the amount of promoter methylation in a control sample, wherein
when the amount of promoter methylation on at least one or more DNA
target sites is less than the amount of promoter methylation in the
control sample, the patient is diagnosed as having HCC.
[0033] It will be understood by those of ordinary skill, that a
diagnosis of HCC can be made by detection of increased methylation
of RASSF1A and/or SSBP2 and/or B4GALT1. Examples of subjects
suspected of having HCC can include, for example, subjects being
chronically infected with Hepatitis B virus, Hepatitis C virus, and
subjects suffering from chronic alcoholism.
[0034] In yet another embodiment, the present invention provides a
method for identifying whether a subject has an increased risk of
GC comprising a) obtaining a biological sample of the gastric
mucosa comprising DNA from a subject, b) determining the amount of
2'-deoxycytidine (2dc) and 5-methyl-2'-deoxycytidine (5mdc) in the
DNA sample, c) generating a global DNA methylation index (GDMI) for
the sample by comparing the amount of 5mdc relative to the amount
of 5mdc+2dc in the sample and generating a ratio, and d) comparing
the GDMI of the sample to the GDMI of a control, wherein when the
GDMI of the sample is less than the GDMI of the control, then the
subject has an increased risk of GC.
[0035] In accordance with one or more embodiments of the present
invention, it will be understood that the types of cancer diagnosis
which may be made, using the methods provided herein, is not
necessarily limited. For purposes herein, the cancer can be any
cancer. As used herein, the term "cancer" is meant any malignant
growth or tumor caused by abnormal and uncontrolled cell division
that may spread to other parts of the body through the lymphatic
system or the blood stream. The cancer can be any cancer, including
any of acute lymphocytic cancer, acute myeloid leukemia, alveolar
rhabdomyosarcoma, adenocarcinoma, bone cancer, brain cancer, breast
cancer, cancer of the anus, anal canal, or anorectum, cancer of the
eye, cancer of the intrahepatic bile duct, cancer of the joints,
cancer of the neck, gallbladder, or pleura, cancer of the nose,
nasal cavity, or middle ear, cancer of the oral cavity, cancer of
the vulva, chronic lymphocytic leukemia, chronic myeloid cancer,
colon cancer, esophageal cancer, cervical cancer, gastrointestinal
carcinoid tumor. Hodgkin lymphoma, hypopharynx cancer,
hepatocellular cancer, kidney cancer, larynx cancer, liver cancer,
lung cancer, malignant mesothelioma, melanoma, multiple myeloma,
nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,
pancreatic cancer, peritoneum, omentum, and mesentery cancer,
pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g.,
renal cell carcinoma (RCC)), small intestine cancer, soft tissue
cancer, stomach cancer, testicular cancer, thyroid cancer, ureter
cancer, and urinary bladder cancer.
[0036] The cancer can be an epithelial cancer. As used herein the
term "epithelial cancer" refers to an invasive malignant tumor
derived from epithelial tissue that can metastasize to other areas
of the body, e.g., a carcinoma. Preferably, the epithelial cancer
is breast cancer. Alternatively, the cancer can be a non-epithelial
cancer, e.g., a sarcoma, leukemia, myeloma, lymphoma,
neuroblastoma, glioma, or a cancer of muscle tissue or of the
central nervous system (CNS).
[0037] The cancer can be a non-epithelial cancer. As used herein,
the term "non-epithelial cancer" refers to an invasive malignant
tumor derived from non-epithelial tissue that can metastasize to
other areas of the body.
[0038] The cancer can be a metastatic cancer or a non-metastatic
(e.g., localized) cancer. As used herein, the term "metastatic
cancer" refers to a cancer in which cells of the cancer have
metastasized, e.g., the cancer is characterized by metastasis of a
cancer cells. The metastasis can be regional metastasis or distant
metastasis, as described herein. Preferably, the cancer is a
metastatic cancer.
[0039] The phrase "controls or control materials" refers to any
standard or reference tissue or material that has not been
identified as having cancer. The GDMI is calculated by determining
the amount of 2'-deoxycytidine (2dc) and 5-methyl-2'-deoxycytidine
(5mdc) in the unknown DNA sample and comparing the amount of 5mdc
relative to the amount of 5mdc+2dc in the sample and generating a
ratio. This is then compared to the GDMI of a control sample.
[0040] The nucleic acids used as primers in embodiments of the
present invention can be constructed based on chemical synthesis
and/or enzymatic ligation reactions using procedures known in the
art. See, for example, Sambrook et al. (eds.), Molecular Cloning, A
Laboratory Manual, 3.sup.rd Edition, Cold Spring Harbor Laboratory
Press, New York (2001) and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and John Wiley
& Sons, NY (1994). For example, a nucleic acid can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed upon hybridization (e.g., phosphorothioate
derivatives and acridine substituted nucleotides). Examples of
modified nucleotides that can be used to generate the nucleic acids
include, but are not limited to, 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguaninc,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-substituted adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.
Alternatively, one or more of the nucleic acids of the invention
can be purchased from companies, such as Macromolecular Resources
(Fort Collins, Colo.) and Synthegen (Houston, Tex.).
[0041] The nucleotide sequences used herein are those which
hybridize under stringent conditions preferably hybridize under
high stringency conditions. By "high stringency conditions" is
meant that the nucleotide sequence specifically hybridizes to a
target sequence (the nucleotide sequence of any of the nucleic
acids described herein) in an amount that is detectably stronger
than non-specific hybridization. High stringency conditions include
conditions which would distinguish a polynucleotide with an exact
complementary sequence, or one containing only a few scattered
mismatches from a random sequence that happened to have a few small
regions (e.g., 3-10 bases) that matched the nucleotide sequence.
Such small regions of complementarity are more easily melted than a
full-length complement of 14-17 or more bases, and high stringency
hybridization makes them easily distinguishable. Relatively high
stringency conditions would include, for example, low salt and/or
high temperature conditions, such as provided by about 0.02-0.1 M
NaCl or the equivalent, at temperatures of about 50-70.degree.
C.
[0042] As used herein, the term "host cell" refers to any type of
cell that can contain the viral DNA disclosed herein. The host cell
can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or
can be a prokaryotic cell, e.g., bacteria or protozoa. The host
cell can be a cultured cell or a primary cell, i.e., isolated
directly from an organism, e.g., a human. The host cell can be an
adherent cell or a suspended cell, i.e., a cell that grows in
suspension. Suitable host cells are known in the art and include,
for instance, DH5.alpha. E. coli cells, Chinese hamster ovarian
cells, monkey VERO cells, COS cells, BC-3 cells, and the like. In
an embodiment, the host cell is preferably a mammalian cell. Most
preferably, the host cell is a human cell or human cell line. The
host cell can be of any cell type, can originate from any type of
tissue, and can be of any developmental stage.
[0043] The term "isolated and purified" as used herein means a
protein that is essentially free of association with other proteins
or polypeptides, e.g., as a naturally occurring protein that has
been separated from cellular and other contaminants by the use of
antibodies or other methods or as a purification product of a
recombinant host cell culture.
[0044] The term "biologically active" as used herein means an
enzyme or protein having structural, regulatory, or biochemical
functions of a naturally occurring molecule.
[0045] The term "reacting" in the context of the embodiments of the
present invention means placing compounds or reactants in proximity
to each other, such as in solution, in order for a chemical
reaction to occur between the reactants.
[0046] As used herein, the term "treat," as well as words stemming
therefrom, includes diagnostic and preventative as well as disorder
remitative treatment.
[0047] As used herein, the term "subject" refers to any mammal,
including, but not limited to, mammals of the order Rodentia, such
as mice and hamsters, and mammals of the order Logomorpha, such as
rabbits. It is preferred that the mammals are from the order
Carnivora, including Felines (cats) and Canines (dogs). It is more
preferred that the mammals are from the order Artiodactyla,
including Bovines (cows) and Swines (pigs) or of the order
Perssodactyla, including Equines (horses). It is most preferred
that the mammals are of the order Primates, Ceboids, or Simoids
(monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human.
[0048] The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or
complete treatment or prevention. Rather, there are varying degrees
of treatment or prevention of which one of ordinary skill in the
art recognizes as having a potential benefit or therapeutic effect.
In this respect, the inventive methods can provide any amount of
any level of diagnosis, screening, or other patient management,
including treatment or prevention of cancer in a mammal.
Furthermore, the treatment or prevention provided by the inventive
method can include treatment or prevention of one or more
conditions or symptoms of the disease, e.g., cancer, being treated
or prevented. Also, for purposes herein, "prevention" can encompass
delaying the onset of the disease, or a symptom or condition
thereof.
EXAMPLES
[0049] Gastric tissue samples and DNA extraction. A total of 402
samples were collected at the National Institute de Neoplasicas
(NEO) and the National Hospital Arzobispo Loayza (LOY) in Lima, Per
, from 75 gastric cancer patients (case study group) and 126
individuals diagnosed with gastritis (control study group). Two
sample biopsies were taken from each patient (201 patients in total
for both study groups, mean age of 62.5.+-.15.1 years, age range
18-88 years). Informed written consent was obtained from all
patients included in the study and research protocols were approved
by Institutional Review Boards of the National Institute de
Neoplasicas, the National Hospital Arzobispo Loayza, the
Universidad Peruana Cayetano Heredia and the Johns Hopkins
University School of Medicine.
[0050] The criteria for a patient's inclusion in the study were to
either have gastro-duodenal symptoms or a suspicion of gastric
cancer. Patients came to the clinic referring symptoms including,
for example, heartburn (81.8%), belching (76.4%), distension
(74.2%), abdominal pain (51.1%), nausea (39.6%) and acid
regurgitation (26.3%). Biopsies were obtained from the cancer
lesion and from the gastric antrum for the controls. Tissue was
then fixed in 10% formalin buffer and embedded in paraffin for
microscopy histological examination. Hematoxylin and eosin-stained
histological slides were scored using the Sydney System. Biopsies
indicative of intestinal metaplasia or H. pylori infection were
also stained with PAS. All the neoplastic tissue used in this study
was classified as gastric adenocarcinoma, and was confirmed by
histopathology.
[0051] DNA from the tissue samples was extracted using the QIAmp
DNA Mini Kit (QIAGEN, Germany) and stored at -20.degree. C. until
use. DNA concentrations were measured using Nanodrop ND-1000
spectrophotometer.
[0052] Gastric global methylation analysis. Global DNA methylation
levels were determined by ELISA using the MDQI, Imprint.TM.
Methylated DNA Quantification Kit (Sigma, St. Louis, Mo.) according
to manufacturer's instructions. Quantification was based on the
estimation of GDMI that was obtained by a single point method. The
differential methylation levels of the samples were relative to the
methylated control DNA. Each analysis for the blank, sample(s) and
control DNA had been performed in duplicate, and the average of the
absorbance readings at 450 nm (A.sub.450) was used for
calculations. The global DNA methylation level for each sample was
calculated according to the equation:
[(A.sub.450avSample-A.sub.450avBlank)/(A.sub.450avMethylated
Control DNA-A.sub.450avBlank)].times.100.
[0053] HCC Patient selection. De-identified frozen primary HCC,
adjacent non-tumor (cirrhotic and non-cirrhotic), and normal liver
(non-cirrhotic tissue obtained from autopsies) tissue samples were
obtained from the Johns Hopkins University School of Medicine and
the Human Cooperative Tissue Network. The study protocol conforms
to the ethical guidelines of the 1975 Declaration of Helsinki as
reflected in a priori approval by the Johns Hopkins Institutional
Review Board. All patients had not undergone therapy prior to
sample collection. The samples were frozen in liquid nitrogen and
stored in -80.degree. C.
[0054] HCC DNA extraction, bisulfite conversion and MeDIP
enrichment. Tissue samples were digested with 1% SDS and 50
.mu.g/ml proteinase K (Bushranger Mannheim) at 48.degree. C.
overnight, followed by phenol/chloroform extraction and ethanol
precipitation of DNA as previously described. Prior to using QMSP
and the Illumina BeadChip assay bisulfite modification of 2 ug of
genomic DNA was performed as previously described (J. Clin. Oncol.,
2005, 23(27):6569-75). Prior to using the Nimblegen tiling arrays
500 ng of genomic DNA was sheared using a water bath sonicator
(Bioruptor UCD-200, Diagenode). Sonicated DNA was then analyzed on
a 1.5% agarose gel to ensure that it had an optimal size of
200-1000 bp. MeDIP was subsequently performed with the Methyl DNA
Immunoprecipitation Kit (Epigentek). Fractions of Input DNA and
Immunoprecipitated DNA from each sample were subsequently sent to
Nimblegen for labeling, hybridization and scanning.
[0055] Illumina BeadChip array. Bisufite treated DNA from 3 HCC
samples and 3 adjacent normal liver samples was hybridized to the
HumanMethylation 27K BeadChip, which quantitatively interrogates
27.578 CpG loci covering more than 14,000 genes at
single-nucleotide resolution. The Infinium Methylation assay
detects cytosine methylation at CpG islands based on highly
multiplexed genotyping of bisulfite-converted genomic DNA (gDNA).
The assay interrogates these chemically differentiated loci using
two site-specific probes, one designed for the methylated locus (M
bead type) and another for the unmethylated locus (U bead type).
Single-base extension of the probes incorporates a labeled ddNTP,
which is subsequently stained with a fluorescence reagent. The
level of methylation for the interrogated locus can be determined
by calculating the ratio of the fluorescent signals from the
methylated vs. unmethylated sites.
[0056] Nimblegen 385K CpG Island Plus Promoter Array. DNA (500 ng)
from 3 liver tissue samples (1 HCC and 2 non-cirrhotic normal liver
samples) enriched with MeDIP were hybridized to Nimblegen Promoter
plus CpG Island 385K oligonucleotide tiling arrays. A single array
design covers 28,226 CpG islands and promoter regions for 17.000
RefSeq genes. The promoter region covered is 1 kb long: 800 bp
upstream from the Transcription Start Site and 200 bp downstream
from the transcription start site. Small CpG islands are extended
at both ends for a total additional coverage of 700 bp for more
reliable detection. DNA methylation positive control regions, such
as the HoxA gene cluster, H19/IGF2 cluster, KCNQ1 cluster, and
IGF2R gene, are also included on the array.
[0057] Bioinformatics analysis of methylation array data. The
Microarray Core at Johns Hopkins School of Medicine performed the
bioinformatics analysis of the Infinium array data using Illumina's
proprietary BeadStudio.TM. software package to provide average
methylation Beta values for each probe. Nimblegen performed the
bioinformatics analysis for the 385K CpG Island Plus Promoter
Array. Nimblegen uses the ACME algorithm to identify
hypermethylated genes that have a statistically significant
methylation peak score above 2 (Methods Enzymol., 2006,
411:270-82).
[0058] Gene selection from public databases of known methylation
events in cancer. Candidate gene selection for promoter methylation
analysis was performed utilizing existing databases of known
methylation events in cancer (BMC Bioinformatics, 2008, 9:22;
Nucleic Acids Res., 2008, 36(Database issue):D836-41). We generated
a list of genes that have been previously shown to be
hypermethylated in HCC and in other tumor tissues. We ranked the
list by the frequency in which the genes had been identified in
different studies. From this list we choose three genes to develop
QMSP primers and probes that examine an 800 bp region upstream from
the transcription start site using the following inclusion
criteria. One of the genes, sequence-specific single-stranded
DNA-binding protein 2 (SSBP2) was shown to be hypermethylated in
other solid tumors. A second gene, beta-1,4-galactosyltransferase-1
(B4GALT1), has not been previously shown to be hypermethylated in
cancer. The third gene, Ras association domain family member 1
(RASSF1A), was shown to be hypermethylated in HCC.
[0059] Quantitative Methylation Specific PCR. DNA from 27 HCC and
21 adjacent normal tissue samples (cirrhotic, non-cirrhotic and
cryptogenic) was bisulfite treated and analyzed with QMSP.
Fluorogenic PCR reactions were carried out in a reaction volume of
20 .mu.L consisting of 600 nmol/l of each primer; 200 .mu.mol/l
probe; 0.75 units platinum Taq polymerase (Invitrogen); 200
.mu.mol/1 of each dATP, dCTP, dGTP, and dTTP; 200 nmol/l ROX dye
reference (Invitrogen); 16.6 mmol/1 ammonium sulfate; 67 mmol/l
Trizma (Sigma, St. Louis, Mo.): 6.7 mmol/l magnesium chloride; 10
mmol/l mercaptoethanol; and 0.1% DMSO. Duplicates of three
microliters (3 .mu.l) ofbisulfite-modified DNA solution were used
in each real-time methylation-specific PCR (QMSP) amplification
reaction. Primers and probes were designed to specifically amplify
the promoters of the three genes of interest (RASSF1A, SSBP2 and
B4GALT1) and the promoter of a reference gene, actin-B (ACTB).
Primer and probe sequences and annealing temperatures are provided
in FIG. 4.
[0060] Amplification reactions were carried out in 384-well plates
in a 7900HT Fast Real-Time PCR System (Applied Biosystems) and were
analyzed by SDS 2.2.1 Sequence Detector System (Applied
Biosystems). Thermal cycling was initiated with a first
denaturation step at 95.degree. C. for 3 minutes, followed by 40
cycles of 95.degree. C. for 15 seconds and 58.degree. C. for 1
minute. Each plate included patient DNA samples, positive
(Bisulfite-converted Universal Methylated Human DNA Standard (Zymo
Research)) and negative (normal leukocyte DNA or DNA from a known
unmethylated cell line) controls, and multiple water blanks. Serial
dilutions (60 ng, 6 ng, 0.6 ng, 0.06 ng and 0.006 ng) of
Bisulfite-converted Universal Methylated Human DNA Standard (ZYMO
RESEARCH.TM.) were used to construct a standard curve for each
gene.
[0061] Statistical analysis for QMSP and methylation array data.
QMSP values were adjusted for DNA input by expressing results as
ratios between 2 absolute measurements. The relative level of
methylated DNA for each gene in each sample was determined as a
ratio of QMSP for the amplified gene to ACTB and then multiplied by
100 for easier tabulation ((average DNA quantity of methylated gene
of interest/average DNA quantity for internal reference gene
b-actin).times.100). The samples were categorized as unmethylated
or methylated based on detection of methylation above a threshold
set for each gene. For quality control, all amplification curves
were visualized and scored without knowledge of the clinical data.
ROC curves were used to identify a cut-off ratio above the highest
control ratio observed for each gene to set specificity at 100%.
Hypermethylation ratios for each gene were compared between cancer
HCC and non-HCC samples. Once the best individually discriminating
genes were found, 2-gene, 3-gene panels were tested to identify the
highest sensitivity with specificity set at 100% for each gene.
[0062] Statistical analysis. All data were calculated using the
commercially available software package, STATA/IC 10 (Statacorp.
Texas. USA) and results with a p<0.05 were considered as
statistically significant. Student's t-test or ANOVA test was used
for analyzing distributions or variances respectively.
Example 1
[0063] Patients' characteristics and design of the GC case control
study. Between March 2004 and June 2010, a total of 201 patients
met the eligible criteria and were included in our study. Utilizing
the updated Sydney system for the classification of gastritis, 25
cases clinically diagnosed as cancer, were characterized as
gastritis by pathologists at two separate institutions. Thus a
second study cohort had been introduced, consisted of 50 patients
in the case study group and 151 patients in the control study
group. Table 1 summarizes the pathological characteristics of the
patients comprising the control study group.
TABLE-US-00001 TABLE 1 Classification of control study group
(gastritis patients) according to pathological characteristics.
Controls Superficial Depth Histology Inflammation Inflammation
Level of inflammation Mild 41 18 Moderate 33 34 Severe 12 12
n/d.sup.a 1 Atrophy Negative 85 40 Positive 2 24 Intestinal
Metaplasia Negative 83 34 Positive 4 30 Helicobacter Pylori
Negative 33 36 Postive 54 27 n/d.sup.a 1 .sup.aNo available
data
Example 2
Evaluation of GDMI and Statistical Analysis and Discrimination
Between Control and Case Study Group
[0064] The control cases found to possess an increased prevalence
of global DNA methylation whereas the case study group had a global
hypomethylation profile. The mean GDMI for the control study group
was estimated to 5.7 (95% CI, 4.93-6.41) and the mean GDMI for the
case study group was 3.7 (95% CI, 2.99-4.39), providing a
statistical significant discrimination between the two groups
(p=0.0016) (FIG. 1).
Example 3
Evaluation of GDMI According to Histological Classification in
Control Study Group
[0065] The statistical analysis of the control study group was
separated into two parts, according to the grade of inflammation.
First DNA methylation levels were measured among all gastritis
patients to distinguish those with superficial inflammation, and
deep inflammation. Subsequently, the patients with deep
inflammation were discriminated by pathological
characteristics.
[0066] Among all gastritis patients, a statistically significant
difference (p=0.02) was found in the GDMI between patients with
superficial (mean GDMI=6.4, 95% CI, 5.34-7.5) and deep inflammation
(mean GDMI=4.7, 95% CI, 3.74-5.56). Samples from gastritis patients
with superficial inflammation were found to be more frequently
methylated when compared to samples from gastritis patients with
deep inflammation (FIG. 2).
Example 4
[0067] Among the gastritis patients with deep inflammation, a
significant difference was found in the GDMI when comparing samples
that were negative and positive for intestinal metaplasia, with the
later presenting lower global methylation levels (p=0.03, mean
GDMI=5.5, 95% CI, 3.98-6.93 in negative cases and mean GDMI=3.7,
95% CI, 2.75-4.73 in positive ones) (FIG. 3).
Example 5
[0068] Patient characteristics of the liver cancer case control
study can be seen in FIG. 5. The majority (58%) of the patients in
our study were men. The mean age of the patients in this study was
47.3 years, and most of (56%) of patients were over 50 years old.
The ethnicity of the patients in our study was White (74%), Black
(23%) and Asian (2%). The most frequent HCC risk factor seen in the
patients of this study was viral infection with HCV (35%) or HBV
(5%). Interestingly cryptogenic cirrhosis was seen in 26% of the
patients. Alcohol intake was the risk factor for a handful of
patients (5%).
Example 6
[0069] Global and gene specific differential DNA promoter
methylation arrays. Scatterplots were used to compare global and
gene specific promoter DNA methylation values between HCC normal
liver tissue samples hybridized to the 385K Nimblegen tiling array
after DNA enrichment with MeDIP (MeDIP-chip). FIG. 6 shows
representative scatterplots and histograms in which a decrease in
global DNA promoter methylation clearly distinguishes between HCC
and normal tissue. Scatterplots and histograms of promoter-wide DNA
methylation array data provide a snapshot of the differences in
methylation patterns between tumor and normal samples.
Promoter-wide promoter hypomethylation was observed in the tumor
when compared to normal samples. The representative tumor sample
has less significant methylated probes (1,503) than either one of
the normal liver tissue samples (2,585 and 2,2887 respectively).
Furthermore, the median methylation score was significantly lower
for the tumor sample (5.7) than for the normal samples (6.7).
[0070] Unsupervised clustering was used to create heat maps,
comparing gene specific methylation between HCC samples and
adjacent normal liver samples (data not shown).
Example 7
[0071] Promoter hypermethylation in tumor and adjacent normal
samples. A search of publicly available methylation databases found
a combined total of 549 methylated genes when searching for
hepatocellular carcinoma (389) and hepatoma (160), 451 of which
were unique genes. Promoter methylation of 3 genes was then
quantified. One gene was already found to be hypermethylated in HCC
by several groups (RASSF1A), and two genes that were found to be
methylated in other tumors but not in HCC (B4GALT 1 and SSBP2). The
promoter methylation of these 3 genes were quantified in 27 HCC
samples and 20 adjacent normal samples. To determine the frequency
of methylation primers and probes were used for QMSP previously
designed in our laboratory based on bisulfite sequencing data. FIG.
4 provides the primer and probe sequences for these three genes.
Area under the curve was calculated using known methods (N. Engl.
J. Med., 2007. 357(16):1589-97).
Example 8
[0072] RASSF1A was methylated in 14/27 (52%) of HCC samples and in
1/17 (6%) of adjacent normal samples. B4GALT1 was methylated in
14/27 (52%) of HCC samples and in 0/20 (0%) of adjacent normal
samples. SSBP2 was methylated in 14/27 (52%) of HCC samples and in
6/18 (33%) of adjacent normal samples. Most of the HCC samples
(78%) had at least one of these three genes methylated, while less
than half of the adjacent normal samples (44%) had one gene
methylated. Methylation of at least two of these genes was observed
in 70% of the HCC samples and in 0% of the adjacent normal samples
(FIG. 7).
Example 9
[0073] ROC curves were used to determine the sensitivity and
specificity of the three genes individually and combined in a
biomarker panel (FIG. 8A) RASSF1A methylation in the examined
tissue samples had a sensitivity of 52%, a specificity of 100% and
an AUC of 0.73 (95% CI, 0.57-0.88). B4GALT1 methylation in the
examined tissue samples had a sensitivity of 52%, a specificity of
100% and an AUC of 0.75 (95% CI, 0.71-0.89). SSBP2 methylation in
the examined tissue samples had a sensitivity of 38%, a specificity
of 100% and an AUC of 0.58 (95% CI, 0.40-0.75) (Table 2).
[0074] When the methylation status of these three genes was
included in a logistic regression model together with gender, age
and etiology the sensitivity was 87%, the specificity was 100% and
the AUC was 0.91 (FIG. 8B)
[0075] The methods of the present invention provide promoter-wide
and gene-specific methylation platforms that interrogate the
promoter region can be used to distinguish between HCC and non-HCC
tissue.
[0076] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0077] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0078] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
12125DNAHomo sapiens 1tggtgatgga ggaggtttag taagt 25227DNAHomo
sapiens 2aaccaataaa acctactcct cccttaa 27318DNAHomo sapiens
3gcgttgaagt cggggttc 18424DNAHomo sapiens 4cccgtacttc gctaacttta
aacg 24519DNAHomo sapiens 5taggaaacgg gtttcgacg 19619DNAHomo
sapiens 6ccgtccactt tctttaccg 19720DNAHomo sapiens 7atttttgcgg
tcgtagcggt 20821DNAHomo sapiens 8ttctacgaca aatctaacga a
21930DNAArtificial Sequencesynthetic 9accaccaccc aacacacaat
aacaaacaca 301024DNAArtificial Sequencesynthetic 10acaaacgcga
accgaacgaa acca 241125DNAArtificial Sequencesynthetic 11cgttaaacaa
cgaaatccaa ccgaa 251224DNAArtificial Sequencesynthetic 12atatccaaaa
cgccgcgaaa ctcc 24
* * * * *