U.S. patent application number 09/759917 was filed with the patent office on 2001-07-12 for methods and kits for diagnosing and determination of the predisposition for diseases.
Invention is credited to Feinberg, Andrew P..
Application Number | 20010007749 09/759917 |
Document ID | / |
Family ID | 22357640 |
Filed Date | 2001-07-12 |
United States Patent
Application |
20010007749 |
Kind Code |
A1 |
Feinberg, Andrew P. |
July 12, 2001 |
Methods and kits for diagnosing and determination of the
predisposition for diseases
Abstract
The present invention provides a method and a kit for the
purpose of diagnosing a disease or determining the predisposition
for a disease by measuring abnormalities in imprinting of a gene or
population of genes. The disease that can be diagnosed by the
present invention is selected from any disease that is mediated by,
or is associated with, a particular gene or combination of genes
that are subject to imprinting. According the present invention,
the imprinting can be abnormally on or can be abnormally off. In
those cases where the particular gene that is being examined is
normally imprinted, but in the disease state is abnormally not
imprinted, the present invention is designed to detect the "loss of
imprinting" (hereinafter "LOI") thereby indicating that the disease
may be present.
Inventors: |
Feinberg, Andrew P.;
(Lutherville, MD) |
Correspondence
Address: |
KILPATRICK STOCKTON LLP
2400 MONARCH TOWER
3424 PEACHTREE ROAD, NE
ATLANTA
GA
30326
US
|
Family ID: |
22357640 |
Appl. No.: |
09/759917 |
Filed: |
January 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09759917 |
Jan 12, 2001 |
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09114825 |
Jul 14, 1998 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
A61K 31/506 20130101;
A61K 31/685 20130101; C12Q 1/6809 20130101; A61K 31/19 20130101;
A61K 31/7068 20130101; C12Q 2600/154 20130101; C12Q 2600/172
20130101; A61K 31/235 20130101; C12Q 1/6827 20130101; A61K 31/215
20130101; A61K 31/00 20130101; A61K 31/165 20130101; C12Q 1/6886
20130101; A61K 31/136 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Goverment Interests
[0001] This invention was made with Government support under grants
from the National Institutes of Health. The United States
Government may have certain rights in the claimed invention.
Claims
1. A method for detecting the presence of a disease, comprising:
obtaining a tissue sample from a subject; and screening said tissue
sample for abnormal imprinting in at least one gene.
2. The method of claim 1, wherein the disease is cancer.
3. The method of claim 2, wherein the cancer is colorectal cancer,
esophageal cancer, stomach cancer, leukemia/lymphoma, lung cancer,
prostate cancer, uterine cancer, breast cancer, skin cancer,
endocrine cancer, urinary cancer, pancreatic cancer, other
gastrointestinal cancer, ovarian cancer, cervical cancer, head
cancer, neck cancer, or adenomas.
4. The method of claim 2, wherein said cancer is colorectal
cancer.
5. The method of claim 2, wherein said at least one gene is IGF2,
H19, p57.sup.KIP2, KvLQT1, TSSC3, TSSC5, or ASCL2.
6. The method of claim 2, wherein said at least one gene is
IGF2.
7. The method of claim 2, wherein said tissue sample is cells from
blood, colon, colonic mucosa.
8. The method of claim 2, comprising measuring the degree of LOI
for at least one gene in said tissue sample.
9. A method for assessing the risk of contracting cancer,
comprising: obtaining a tissue sample from a subject; and screening
said tissue sample for loss of imprinting in at least one gene.
10. The method of claim 8, wherein said cancer is colorectal
cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, lung
cancer, prostate cancer, uterine cancer, breast cancer, skin
cancer, endocrine cancer, urinary cancer, pancreatic cancer, other
gastrointestinal cancer, ovarian cancer, cervical cancer, head
cancer, neck cancer, or adenomas.
11. The method of claim 9, wherein said cancer is colorectal
cancer.
12. The method of claim 9, wherein said at least one gene is IGF2,
H19, p57.sup.KIP2, KvLQT1, TSSC3, TSSC5, or ASCL2.
13. The method of claim 9, wherein said at least one gene is IGF2.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to methods for detecting
and/or screening for the presence of diseases such as cancer, and
for assessing the risk of contracting a disease. The present
invention also relates to methods for detecting and/or screening
for the presence of DNA microsatellite instability.
BACKGROUND OF THE INVENTION
[0003] The single greatest impediment to cancer diagnosis is the
general requirement that the tumor itself must be detected
directly. Efforts to identify genetic abnormalities in normal
tissues of patients with cancer or at risk of cancer have been
disappointing. For example, BRCA1 mutations are present in only
about 1% of breast cancers. A small fraction of patients with
colorectal cancer have predisposing mutations in the APC gene
(>1%), causing adenomatous polyposis coli. An even smaller
fraction show mutations in genes responsible for replication error
repair (>2% of colon cancer patients, or much less than 1% of
the population), show mutations in genes responsible for nucleotide
mismatch error repair causing hereditary nonpolyposis colorectal
cancer (HNPCC or Lynch syndrome).
[0004] Genetic studies of colorectal cancer present a paradox, in
that 15-40% of sporadically occurring tumors show DNA
microsatellite instability, depending on the number of
microsatellite markers that are used to detect it, even though the
overwhelming majority of such tumors do not show mutations in known
error repair genes. Furthermore, microsatellite instability in many
common tumors, including those of the stomach, colon, and lung, is
associated with a younger age, positive family history, and/or less
accessible and detectable location, suggesting that a relatively
large subgroup of cancer patients in the general population are at
increased risk of cancer, even though they do not fall within a
well-defined syndrome.
[0005] Microsatellite instability, in particular, requires for
identification that a patient already have a tumor, because the
assay compares microsatellite marker length between the monoclonal
tumor cell population and normal tissue derived from the same
patient. Most importantly, family history still remains the most
reliable diagnostic procedure for identifying patients at risk of
cancer. A molecular diagnostic approach that might identify
patients with cancer or at risk of cancer, using only normal
tissue, would offer a decisive advantage for intervention and
treatment.
[0006] Thus, there remains a need for a diagnostic method for
detecting and/or screening for the presence of diseases and/or the
risk of contracting a disease. In particular, there remains a need
for a method for detecting and/or screening for the presence of
cancer, which does not require a tumor sample. There also remains a
need for a method of detecting and/or screening for the presence of
cancer and/or the risk of contracting cancer which can be applied
to a wide section of the population. There also remains a need for
a method of detecting and/or screening for the presence of
replication error repair defects which does not require a tumor
sample. There also remains a need for a method for quantifying the
degree of loss of imprinting. There also remains a need for a
method for screening infants for the risk of sudden infant death
syndrome (SIDS). There-also remains a need for kits for carrying
out these methods.
SUMMARY OF THE INVENTION
[0007] The present invention provides a method and a kit for the
purpose of diagnosing a disease by measuring abnormalities in
imprinting of a gene or population of genes. The disease that can
be diagnosed by the present invention is selected from any disease
that is mediated by, or is associated with, a particular gene or
combination of genes that are subject to imprinting. According the
present invention, the imprinting can be abnormally on or can be
abnormally off or partially abnormally on or off. In those cases
where the particular gene that is being examined is normally
imprinted, but in the disease state is abnormally not imprinted,
the present invention is designed to detect the "loss of
imprinting" (hereinafter "LOI") thereby indicating that the disease
may be present.
[0008] The diseases that can be diagnosed according to the present
invention includes, but is not limited to, cancers, SIDS, birth
defects, mental retardation, diabetes & gestational diabetes.
Cancers that can be diagnosed according to the present invention
include, but are not limited to, colorectal cancer, esophageal
cancer, stomach cancer, leukemia/lymphoma, lung cancer, prostate
cancer, uterine cancer, breast cancer, skin cancer, endocrine
cancer, urinary cancer, pancreatic cancer, other gastrointestinal
cancer, ovarian cancer, cervical cancer, head cancer, neck cancer,
and adenomas.
[0009] The present invention also provides a method and a kit for
determining if a patient is predispositioned to a particular
disease. In this case, normal tissue can be tested to determine
whether there is abnormal imprinting for a particular gene or
combination of genes. If the imprinting is abnormal, e.g., either
abnormally on or abnormally off, then the patient may have a
predisposition for a particular disease. Appropriate screening of
other factors can then be done a periodic intervals to be able to
detect the disease early.
[0010] Because the present invention is particularly useful for
determining if a patient is predispositioned for a particular
disease, such as cancer, the present invention is particularly
useful for screening populations of individuals to determine if
individuals are predispositioned for a particular disease. By
having the capability of efficiently screening a large population
of individuals for a particular disease, e.g., colon cancer,
individuals that have the cancer in the early stages or individuals
that are predispositioned for the cancer can be identified early
and appropriate treatment can be initiated.
[0011] According to the present invention, the testing of an
individual to determine if imprinting of a gene is normal or
abnormal can be done on a wide variety of tissues. For example, in
a test for colorectal cancer, one does not have to test tissue from
the colon, but can test tissue from other parts of the body,
including, but not limited to, blood cells, skin, hair, etc.
Therefore, the present invention provides a method of testing any
tissue in the body for a disease that is specific for a particular
tissue in the body.
[0012] Analysis of imprinting of genes of biological tissues and
cells to be used for transplantation can be performed according to
the present invention to avoid the possibility of increasing a
disease risk in the transplant recipient. Examples of tissues or
cells that can be analyzed according to the present invention
includes, but is not limited to, skin grafts, ligaments, eyes,
kidney, liver, heart valves, lung, bone-marrow (Leukemia), neural
tissue-embryonic neural tissue (used for variety of purposes such
as increase dopamine production in Parkinson's patients, increase
acetylcholine production in Alzheimer's patients, for enhancement
of plasticity (norepinephrine), increase production of hypothalamic
hypophysiotropic factors, development of neocortical cells, motor
neurons, sensory neurons, blood-white cells and others of myelocyte
lineage, muscle-myoblasts, seeding of "blast cells," and
genetically engineered cells for administration to patients.
[0013] According to one embodiment of the present invention, there
is a strong correlation between the presence or absence of LOI in a
subject's somatic cells and the presence of disease and/or the risk
of contracting a disease. There is a strong correlation between the
presence of LOI in a subject's somatic cells and the presence of
replication repair error defects. There is also a strong
correlation between the degree of LOI present in a subject's
somatic cells and the presence of cancer and/or the risk of
contracting cancer. In addition, there is a strong correlation
between the degree of LOI present in a subject's somatic cells and
the degree of replication repair error defects present in a
subject's somatic cells. There is a strong correlation between the
degree of LOI present in a subject's somatic cells and the survival
rate of the subject upon contracting cancer. According to the
present invention, the general population, in particular infants,
may be screened for the risk of genetic diseases, in particular
SIDS, by examining their imprinting patterns.
[0014] Accordingly, it is an object of the present invention to
provide novel methods for detecting or determining a predisposition
for the presence of a disease in an individual.
[0015] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
colorectal cancer.
[0016] It is yet another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
stomach cancer.
[0017] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
esophageal cancer.
[0018] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
leukemia.
[0019] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
lung cancer.
[0020] It is yet another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
prostate cancer.
[0021] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
uterine cancer.
[0022] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
breast cancer.
[0023] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
skin cancer.
[0024] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
endocrine cancer.
[0025] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
urinary cancer.
[0026] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
lymphoma.
[0027] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
pancreas cancer.
[0028] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
gastrointestinal cancer.
[0029] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
ovarian cancer.
[0030] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
cervical cancer.
[0031] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
head cancer.
[0032] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
neck cancer.
[0033] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
adenomas.
[0034] It is another object of the present invention to provide
novel methods for detecting and/or screening for the presence of
replication error repair defects.
[0035] It is another object of the present invention to provide
novel methods for assessing the risk of contracting cancer.
[0036] It is another object of the present invention to provide
novel methods for assessing the risk of contracting colorectal
cancer.
[0037] It is another object of the present invention to provide
novel methods for assessing the risk of contracting stomach
cancer.
[0038] It is another object of the present invention to provide
novel methods for assessing the risk of contracting esophageal
cancer.
[0039] It is another object of the present invention to provide
novel methods for assessing the risk of contracting leukemia.
[0040] It is another object of the present invention to provide
novel methods for assessing the risk of--It is another object of
the present invention to provide novel methods for assessing the
risk of contracting prostate cancer.
[0041] It is another object of the present invention to provide
novel methods for assessing the risk of contracting uterine
cancer.
[0042] It is another object of the present invention to provide
novel methods for assessing the risk of contracting breast
cancer.
[0043] It is another object of the present invention to provide
novel methods for assessing the risk of contracting skin
cancer.
[0044] It is another object of the present invention to provide
novel methods for assessing the risk of contracting endocrine
cancer.
[0045] It is another object of the present invention to provide
novel methods for assessing the risk of contracting urinary
cancer.
[0046] It is another object of the present invention to provide
novel methods for assessing the risk of contracting lymphoma.
[0047] It is another object of the present invention to provide
novel methods for assessing the risk of contracting pancreas
cancer.
[0048] It is another object of the present invention to provide
novel methods for assessing the risk of contracting
gastrointestinal cancer.
[0049] It is another object of the present invention to provide
novel methods for assessing the risk of contracting ovarian
cancer.
[0050] It is another object of the present invention to provide
novel methods for assessing the risk of contracting cervical
cancer.
[0051] It is another object of the present invention to provide
novel methods for assessing the risk of contracting head
cancer.
[0052] It is another object of the present invention to provide
novel methods for assessing the risk of contracting neck
cancer.
[0053] It is another object of the present invention to provide
novel methods for assessing the risk of contracting adenomas.
[0054] It is another object of the present invention to provide
novel methods for identifying a subset of the general population
for preventative chemotherapy.
[0055] It is another object of the present invention to provide
novel methods for identifying a subset of the general population
for cancer treatment.
[0056] It is another object of the present invention to provide
novel methods for quantifying the degree LOI.
[0057] It is another object of the present invention to provide
novel methods for determining the prognosis of a patient suffering
from cancer.
[0058] It is another object of the present invention to provide
novel methods for determining the future prognosis, upon
contracting cancer, of a subject who does not yet have cancer.
[0059] It is another object of the present invention to provide
novel methods for screening the general population, in particular
infants, for genetic diseases.
[0060] It is another object of the present invention to provide
novel methods for screening infants for the risk of SIDS.
[0061] It is another object of the present invention to provide
novel kits useful for carrying out such methods.
[0062] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same become better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0064] FIG. 1 schematically shows the strategy for quantitative
analysis of IGF2 imprinting in colon cancer. One of the two
polymorphisms used, an Apa I site in exon 9, is depicted for
illustrating the method. A heterozygous (informative) sample
harbors two alleles, A (without the Apa I site), and B (with the
Apa I site). Heterozygosity of genomic DNA was ascertained by
performing DNA PCR, using primers 3 and 4 across the Apa I site,
and the PCR product was digested with Apa I. Imprinting status was
ascertained by performing RT-PCR on RNA, using primers 1 and 2 in
exons 8 and 9, respectively. The cDNA PCR product, which is shorter
than any possible contaminating genomic DNA product because of
intron splicing, was electrophoresed and purified from an agarose
gel. PCR was then performed using primers 3 and 4, end-labeling one
of the primers. The PCR product was digested with Apa I, analyzed
on a 6% polyacrylamide gel, and quantified on a PhosphorImager. The
B allele is shorter but of equal radioactive intensity to the A
allele. All RT-PCR experiments were performed in parallel in the
presence and absence of reverse transcriptase, from the identical
cDNA product, in order to rule out the presence of contaminating
DNA. The threshold for scoring loss of imprinting (LOI) was less
than a 3:1 ratio between the more abundant and less abundant
alleles. This threshold distinguished both cancers and matched
normal mucosa with microsatellite instability in their tumors. The
quantitative measure of imprinting was reproducible among assays,
and also consistent between paired tumor and normal samples by
paired t-test analysis.
[0065] FIGS. 2A and 2B show genomic imprinting in colorectal cancer
patients; FIG. 2A LOI is seen in both cancer (C) and matched normal
(N) mucosa from patients 1, 2, and 4, who also showed
microsatellite instability in their cancers. Normal imprinting is
seen in both cancer (C) and paired normal (N) mucosa from patients
19 and 21, who did not show microsatellite instability in their
cancers. The A and B alleles are 292 and 229 bp, respectively. FIG.
2B normal imprinting is seen in the normal mucosa of noncancer
patients 1-7. Slight expression of the A allele is seen in patient
3, at a 5.2:1 ratio to the B allele, below the threshold for
scoring LOI.
[0066] FIG. 3 shows the analysis of promoter-specific imprinting in
colon cancer patients. 3(A) Gene-specific cDNA, derived from
reverse transcription with an IGF2 downstream primer, was amplified
using promoter-specific primers. PCR products were subjected to
Southern allele-specific hybridization (SASH) using allele-specific
oligonucleotide probes, as described previously (He et al. (1998)
Oncogene 16:113-119 which is incorporated herein by reference).
Note that promoters 3 and 4 clearly displayed biallelic expression
in all samples examined, while expression from promoters 1 and 2
was not detectable. 3(B) A reconstitution control was performed
concurrently, by mixing cDNA homozygous for the A and B alleles at
varying ratios, and then amplifying with promoter 4-specific
primers. PCR products were detected by allele-specific
oligonucleotide probe A or B respectively. The assay demonstrated
that the amplification of promoter-specific cDNA was linear and
quantitative for both alleles under the conditions used.
[0067] FIG. 4 shows the link between LOI in colorectal cancer. LOI
in matched normal mucosa, and microsatellite instability. Paired
tumor-normal samples show a strong correlation between LOI in the
cancer and LOI in matched normal mucosa, expressed as the ratio of
the more abundant to less abundant allele. LOI also distinguishes
patients with and without MSI in their tumors.
[0068] FIG. 5 shows the loss of imprinting in blood, colonic
mucosa, and tumor of colon cancer patients. LOI is seen in blood
(B), matched normal colonic mucosa (M), and cancer (C) of colon
cancer patients 11, 23, 25, and 27. All four patients showed
microsatellite instability in their tumors.
DETAILED DESCRIPTION
[0069] The present invention provides a novel method for detecting
a disease or measuring the predisposition of a subject for
developing a disease in the future by obtaining a biological sample
from a subject; and screening the biological sample for the
presence of abnormal imprinting. In the present invention, the
subject will typically be a human but also is any organism,
including, but not limited to, a dog, cat, rabbit, cow, bird,
ratite, horse, pig, monkey, etc.
[0070] If the disease to be detected is cancer, the cancer to be
detected or screened includes, but is not limited to, colorectal
cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, lung
cancer, prostate cancer, uterine cancer, breast cancer, skin
cancer, endocrine cancer, urinary cancer, pancreas cancer, other
gastrointestinal cancer, ovarian cancer, cervical cancer, head
cancer, neck cancer, and adenomas. In a particularly preferred
embodiment, the cancer is colorectal cancer.
[0071] The biological sample may be any which is conveniently taken
from the patient and contains sufficient information to yield
reliable results. Typically, the biological sample will be a tissue
sample which contains 1 to 10,000,000, preferably 1000 to
10,000,000, more preferably 1,000,000 to 10,000,000 somatic cells.
However, it is possible to obtain samples which contain smaller
numbers of cells and then enrich the cells. In addition, with
certain highly sensitive assays (e.g., RT-PCR when IGF2 is
abundant, and other methods like DNA methylation even when IGF2 not
abundant) it is possible to get sample size down to single cell
level-with. However, the sample need not contain any intact cells,
so long as it contains sufficient biological material (e.g.,
protein; genetic material, such as DNA or RNA; etc.) to assess the
presence or absence of LOI in the subject.
[0072] According to the present invention, the biological or tissue
sample may be preferably drawn from the tissue which is susceptible
to the type of disease to which the detection test is directed. For
example, the tissue may be obtained by surgery, biopsy, swab,
stool, or other collection method. In addition, it is possible to
use a blood sample and screen either the mononuclear cells present
in the blood or first enrich the small amount of circulating cells
from the tissue of interest, i.e., colon, breast, etc. using a
method known in the art.
[0073] According to one embodiment of the present invention, when
examining a biological sample to detect colorectal cancer, it may
be preferred to obtain a tissue sample from the colon. Such a
tissue sample may be obtained by any of the above described
methods, but the use of a swab or biopsy may be preferred. In the
case of stomach and esophageal cancers, the tissue sample may be
obtained by endoscopic biopsy or aspiration, or stool sample or
saliva sample. In the case of leukemia, the tissue sample is
preferably a blood sample.
[0074] In another preferred embodiment of the present invention,
the biological sample is a blood sample. The blood sample may be
obtained in any conventional way, such as finger prick or
phlebotomy. Suitably, the blood sample is approximately 0.1 to 20
ml, preferably approximately 1 to 15 ml with the preferred volume
of blood being approximately 10 ml.
[0075] In one preferred embodiment, the cancer to be detected is
colorectal cancer, and the biological sample is a tissue sample
obtained from the colon or a stool sample. In another preferred
embodiment, the cancer to be detected is stomach cancer or
esophageal cancer, and the tissue may be obtained by endoscopic
biopsy or aspiration, or stool sample or saliva sample. In another
preferred embodiment, the cancer to be detected is esophageal
cancer, and the tissue is obtained by endoscopic biopsy,
aspiration, or oral or saliva sample. In another preferred
embodiment, the cancer is leukemia/lymphoma and the tissue sample
is blood.
[0076] Genomic imprinting is an epigenetic modification of a
specific parental chromosome in the gamete or zygote that leads to
monoallelic or differential expression of the two alleles of a gene
in somatic cells of the offspring. Imprinting affects various
essential cellular and developmental processes, including
intercellular signaling, RNA processing, cell cycle control, and
promotion or inhibition of cellular division and growth.
[0077] The first deduction of imprinting at the single gene level
involved a transgenic C-myc gene which showed dependence of its
expression on paternal inheritance. The silent maternally inherited
copy was methylated (Swain et al. (1987) Cell 50:719-727).
Disruption of the insulin-like growth factor II (IGF2) gene in
knockout experiments showed that IGF2 is imprinted and expressed
normally only from the paternal allele, and that IGF2 is
biparentally expressed in two neural tissues, the choroid plexus
and the leptomeninges (DeChiara et al. (1991) Cell 64:849-859).
These milestone studies showed that genomic imprinting affects
normal endogenous genes and that imprinting shows tissue-specific
regulation.
[0078] Direct approaches for the identification of novel imprinted
genes include: positional cloning which identifies imprinted genes
near other known imprinted genes (Barlow et al. (1991) Nature
349:84-87); comparing gene expression in parthenogenetic embryos to
that of normal embryos (Kuroiwa et al. (1996) Nat. Genet
12:186-190); and restriction landmark genome scanning (Nagai et al.
(1995) Biochem. Biophys. Res. Commun. 213:258-265).
[0079] Abnormalities of a single gene can affect imprinting of a
proximate genomic region and disrupt multiple disease-causing
genes, the phenotype depending upon the parental origin of the
mutated gene. Two examples of imprinted human disease loci in close
proximity are on chromosome 15. Disrupted imprinting of these loci
is one of the causes of Prader-Willi syndrome (PWS) and Angelman
sypdrome (AS) which involve mental retardation. PWS also causes
obesity, and AS involves gross motor disturbances. Each disorder
can be caused by parental-origin specific uniparental disomy
(Nicholls et al. (1989) Nature 342:281-285; Knoll et al. (1990) Am.
.J. Hum. Genet. 47:149-155) or chromosomal deletions (Knoll et al.
(1989) Am. I. Hum. Genet. 47:149-155; Mattel et al. (1984) Hum.
Genet. 66:313-334). The AS gene has not been isolated to date. PWS
appears to be due to mutations or deletions in the small nuclear
ribonucleoprotein polypeptide N (SNRPN) gene (reviewed in Nicholls
et al. (1993) Curr. Opin. Genet. Dev. 3:445-446). A mutation
affecting splicing of an untranslated upstream exon of SNRPN can
also lead to AS, as well as abnormal imprinting of other loci
(Dittrich et al. (1996) Nat. Genet. 14:163-170).
[0080] It is likely that many more additional imprinted human
disease loci will he identified, because UPD for specific
chromosomes is often associated with multiple congenital anomalies
(Ledbetter, D. H. and Engel, E. (1995) Hum. Mol. Genet.
4:1757-1764). Chromosomes that likely show this phenomenon include
2, 6, 7, 11, 14, 15, 16, 20. and X (Ledbetter, D. H. and Engel, E.
(1995) Hum. Mol. Genet. 4:1757-1764).
[0081] An indirect suggestion of genomic imprinting in cancer came
from investigations of the two embryonic tumors, hydatidiform mole
and complete ovarian teratoma, showing that not only 46 chromosomes
are required to create a normal embryo, but also a balance of
maternal and paternal chromosomes. A relative imbalance leads to
neoplastic growth, and the type of neoplasm depends upon whether
there is a maternal or paternal genetic excess.
[0082] Another tumor apparently associated with imprinting is
familial paraganglioma or glomus tumor. In all cases, the
transmitting parent is the father (Van der Mey et al. (1989) Lancet
2:1291-1294). The gene has recently been localized to 11q22.3-q23
(Heutink et al. (1994) Eur. J. Hum. Genet 2:148-158).
[0083] Loss of heterozygosity (LOH) in the childhood Wilms tumor
occurs on chromosome 11 and the specifically involved region is
11p15 (Reeve et al. (1989) Mol. Cell. Biol. 9:1799-1803). Schroeder
et al. (1987) Am. J. Hum. Genet. 40:413-420, noted that in five of
five cases of LOH, it was the maternal allele that was lost. This
observation has been extended to other tumors of embryonal origin
such as hepatoblastoma and rhabdomyosarcoma (Scrable et al. (1989)
Proc. Nati. Acad. Sci. USA 86:7480-7484).
[0084] The N-myc gene on chromosome 2 shows preferential
amplification of the paternal allele in neuroblastoma (Cheng et al.
(1993) Nature Genet. 4:187-190). Advanced neuroblastoma tumors,
showing N-myc amplification, also show preferential LOH of maternal
chromosome 1, whereas earlier stage tumors without N-myc
amplification do not (Caron et al. (1995) Hum. Mol. Genet
4:535-539). Thus, genetic disturbances involving imprinted genes in
a given type of cancer may involve multiple chromosomes
concurrently. Naumova et al. (1994) Am. J. Hum. Genet. 54:274-281,
found transmission ratio distortion, concordance of 13q loss and
isochromosome 6 of the same parental origin in retinoblastoma,
again consistent with a mechanism of generalized disturbance of
imprinting in embryogenesis leading to increased cancer risk.
[0085] Beckwith-Wiedemann syndrome (BWS) is a disorder of prenatal
overgrowth and cancer, transmitted as an autosomal dominant trait,
or arising sporadically. A clue to a role for genomic imprinting is
increased disease penetrance when BWS is inherited from the mother
(Viljoen, D. and Ramesar, R. (1992)]. Med. Genet. 29:22 1-225). The
tumors of children show preferential loss of maternal lipiS
(Schroeder et al. (1987) Am. J. Hum. Genet. 40:413-420; Scrable et
al. (1989) Proc. Nati. A cad. Sci. USA 86:7480-7484), suggesting
that an imprinted locus could cause BWS and also be involved in
sporadically occurring tumors. Genetic linkage analysis showed that
BWS localizes to 11p13, consistent with this idea of generalized
disruption, and not to 11p13, to which the WT1 gene had been
localized (Ping et al. (1989) Am. J. Hum. Genet. 44:720-723; Koufos
et al. (1989) Am. J. Hum. Genet. 44:711-719).
[0086] More direct evidence for genomic imprinting of 11p15 in BWS
came from studies showing that some patients with BWS have paternal
UPD, involving a region extending from the .beta.-globin locus to
the RAS gene (Henry et al. (1991) Nature 35:609-610). While the
large 10 Mb region does not provide precise localization of an
imprinted gene, it provides a foundation for later studies of
imprinted loci on this chromosome.
[0087] Examination of RNA from Wilms tumor (WT) led to a discovery
that not one but both IGF2 alleles were expressed in 70% of Wilms
tumors (Rainier et al. (1993) Nature 362:747-749; Ohlsson et al.
(1993) Nature Genet. 4:94-97). In addition, in 30% of cases, both
alleles of H19 were expressed (Rainier et al. (1993) Nature
362:747-749). In contrast, examination of RNA from normal tissue
shows normal imprinting with the expression of one allele of IGF2
and H19. This was the first evidence of imprinting in humans. The
term for this novel genetic alteration is loss of imprinting (LOI)
(Rainier et al. (1993) Nature 362:747-749) which simply means loss
of preferential parental origin-specific gene expression and can
involve either abnormal expression of the normally silent allele,
leading to biallelic expression, or silencing of the normally
expressed allele, leading to epigenetic silencing of the locus
(Rainier et al. (1993) Nature 362:747-749; Feinberg (1993) Nature
Genet. 4:110-113; Feinberg et al. (1995) J. Natl. Cancer Inst.
Monographs 17:21-26). Thus, abnormal imprinting in cancer can lead
to activation of normally silent alleles of growth-promoting genes
(Rainier et al. (1993) Nature 362:747-749); (Feinberg (1993) Nature
Genet. 4:110-113); (Feinberg et al. (1995) J Natl. Cancer Inst.
Monographs 17:21-26).
[0088] Subsequently, LOI has been implicated in various tumor
types. At first, LOI was found in other childhood tumors, such as
hepatoblastoma (Rainier et al. (1995) Cancer Res. 55:1836-1838; Li
et al. (1995) Oncogene 11:221-229), rhabdomyosarcoma (Zhan et al.
(1994) J Clin. Invest. 94:445-448), and Ewings sarcoma (Zhan et al.
(1995a) Oncogene 11:2503-2507). LOI of IGF2 and H19 have also now
been found in many adult tumors, including uterine (Vu et al.
(1995) J. Clin. Endocrinol. Metab. 80:16701676 cervical (Doucrasy
12:423430), esophageal (Hibi et ai. (1996) Cancer Res. et al.
(1996) Onco gene 56480-482), prostate (Jartard etai. (1995) Clin.
Cancer Res. 1:14711478), lung cancer (Kondo et ai. (1995) Oncogene
10:1193-1198), choriocarcinoma (Hashimoto et al. (1995) Nat. Genet.
9:109-110), germ cell tumors (Van Gurp et al. (1994) J. Natl.
Cancer Inst. 86:1070-1075) BWS (Steenman et al. (1994) Nature
Genet. 7:433-437) Weksberg et al. (1993) Nature Genet. 5:143-150)
and Wilms tumor (Ogawa et al. (1993) Nature Genet. 5:408-412).
Thus, LOI is one of the most common alterations human cancer.
[0089] Another imprinted gene in 11p15 is p57.sup.kip2 a
cyclin-dependent kinase (CDK) inhibitor similar in its CDK
inhibitory domain to p21.sup.WAF1/CiP1 a target of p53 (Matsuoka et
cii. (1995) Genes Dev. 9:640-662); Lee et al. (1995) Genes. Dev.
9:639-649). The gene was localized within 40 kb of a group of BWS
balanced germline chromosomal rearrangement breakpoints, in
contrast to IGF2 and H19, which are located telomeric to those
breakpoints (Hoovers et al. (1995) Proc. Natl. Acad. Sd. USA 92:
12456-12460). Human p57.sup.kip2 was found to be imprinted with
preferential expression from the maternal allele (Matsuoka ci cii.
(1996) Proc. Natl. A cad. Sci. USA 93:3026-3030). p57.sup.kip2 also
shows abnormal imprinting and epigenetic silencing in some tumors
and BWS patients (Thompson et al. (1996) Cancer Research
56:5723-5727). Other imprinted genes on 11p15 include KvLQTI,
TSSC3, TSSC5, and ASCL2. Abnormalities of one or more of these
genes are implicated in a wide variety of cancers, and birth
defects including BWS SIDS (Schwartz P. J. et al., (1998)
Prolongation of the QT Interval and the Sudden Infant Death
Syndrome, New Engl. J. Med. 338:1709-1714).
[0090] Several lines of evidence provide a role for DNA methylation
in the control of genomic imprinting. First, some imprinted genes
in mice, such as H19, show parental origin-specific,
tissue-independent methylation of CpG islands (Ferguson-Smith et
al. (1993) Nature 362:751-755; Bartolomei et al. (1993) Genes
Develop. 7:1663-1673. This methylation represents imprinting on the
paternal chromosome and is not secondary to changes in gene
expression. Second, knockout mice deficient in DNA
methyltransferase, and exhibiting widespread genomic
hypomethylation, do not show allele-specific methylation of the H19
CpG island and exhibit biallelic expression of H19 and loss of
expression of IGF2 (Li et al. (1993) Nature 366:362-365). Similar
parental origin-specific methylation has also been observed for a
CpG island in the first intron of the maternally inherited,
expressed allele of the IGF2 receptor gene (IGF2R) (Stoger et al.
(1993) Cell 73:61-71). Methyltransferase deficient knockout mice
show loss of methylation of IGF2R and epigenetic silencing of the
gene (Li et al. (1993) Nature 366:362-365).
[0091] Widespread alterations in DNA methylation inhuman tumors
were discovered years ago (Feinberg, A P. (1983) Nature Genet.
4:110-113) and remain the most commonly found alteration in human
cancers. These alterations are ubiquitous to both benign and
malignant neoplasms (Goelz et al. (1985) Science 228:187-190). The
precise role of these changes remain unclear; but, both decreased
and increased methylation have been found at specific sites in
tumors, with an overall decrease in quantitative DNA methylation
(Feinberg et al. (1988) Cancer Res. 48:1159-1161; Feinberg, A. P.
(1988) Prog. Clin. Biol. Res.) 79:309-317; Jones et al. (1990) Adv.
Cancer Res. 54:1-23).
[0092] In humans, as in mice, the paternal allele of a CpG island
in the H 19 gene and its promoter is normally methylated, and the
maternal allele is unmethylated (Steenman et al., (1994) Nature
Genet. 7:433-439; Ferguson-Smith et al. (1993) Nature 362:751-755;
Bartolomei et al. (1993) Genes Develop. 7:1663-1673). Because
tumors with LOI of IGF2 showed reduced expression of H19, the
methylation pattern of H19 has been examined in tumors with LOI. In
all cases showing LOI of IGF2, the H19 promoter exhibits 90%-100%
methylation at the sites normally unmethylated on the maternally
inherited allele (Steenman et al. (1994) Nature 7:433-437; Moulton
et al. (1994) Nature Genet. 7:440-447). Thus, the maternal allele
has acquired a paternal pattern of methylation, consistent with
observed expression of IGF2 on the same maternally derived
chromosome in these tumors. In contrast, tumors without LOI of IGF2
show no change in the methylation of H19, indicating that these
changes are related to abnormal imprinting and not malignancy per
se (Steenman et at. (1994) Nature Genet. 7:433-439; Moulton et at.
(1994) Nature Genet. 7:440-447). The same alterations in
methylation of the maternal allele of H 19 are found in BWS
patients with LOI of IGF2 (Steenman et at. (1994) Nature Genet.
7:433-439; Reik et at. (1994) Hum. Mat. Genet. 3:1297-1301; Reik et
at. (1995) Hum. Mat. Genet. 4:2379-2385).
[0093] A second potential mechanism of LOI may involve disruption
of an imprinting control center on chromosome 11, similar to that
recently described for the BWS/AS region of chromosome 15 (Dittrich
et al. (1996) Nat. Genet. 14: 163-170). A cluster of five BWS
balanced germline chromosomal rearrangement breakpoints lies
between p57.sup.kiP2 on the centromeric side, and IGF2 and H19 on
the telomeric side (Hoovers et al. (1995) Proc. Natl. Acad. Sci.
USA 92:12456-12460). Thus, disruption of a gene spanning this
region could cause abnormal imprinting, as well as BWS and/or
cancer, at least when inherited through the germline.
[0094] Another potential mechanism for LOI involves loss of
trans-acting factors which may establish and maintain a normal
pattern of genomic imprinting once such a pattern is established in
the germline. Trans-acting modifiers of imprinting are likely to
exist, since imprinting of transgenes is host strain-dependent
(Sapienza et al. (1990) Develop. 107:165-168; Allen et al. (1990)
Cell 61:853-361). Such genes might thus act as tumor suppressor
genes in humans and other species.
[0095] Yet another potential mechanism of imprinting that might be
disrupted in cancer involves histone deacetylation which is linked
to X-inactivation in mammals (reviewed in Wolffe, A. P. (1994)
Develop. Genet. 15:463-470) and to telomere silencing in yeast
(Thompson et al. (1994) Nature 369:245-247). Genes for both histone
acetylase and histone deacetylase have recently been isolated
(Brownell et al. (1996) Cell 84:843-851 Taunton et al. (1996)
Science 272:408-411). In addition, telomere silencing in yeast also
involves the action of specific genes, e.g., SIR1-SIR4, some of
which have homologues in mammals (Brachmann et al. (1995) Genes
Develop. 9:2888-2902). Similarly, some examples of gene silencing
in mammals may resemble position-effect variation in Drosophila, a
form of position-dependent epigenetic silencing (Walters et al.
(1996) Genes Develop. 10:185-195). Finally, imprinted loci on
maternal and paternal chromosomes may interact during DNA
replication. Chromosomal regions harboring imprinted genes show
replication and timing asynchrony (Kitsberg et al. (1993) Nature
364:459-463). Furthermore, the two parental homologues of some
imprinted genes show nonrandom proximity in late S-phase (LaSalle.
J. M. and Lalande, M. (1996) Science 272:725-728), suggesting some
form of chromosomal cross-talk, as has been observed for epigenetic
silencing in Drosophila (Tartoff, K. D. and Henikoff, S. (1991)
Cell 65:201-203).
[0096] The human IGF2 and H19 genes are normally imprinted, i.e.,
show preferential expression of a specific parental allele (Rainier
et al. (1993) Nature 362:747-749; Zhang, Y. and Tycko, B. (1992)
Nat. Genet. 1:40-44; Giannoukakis et al. (1993) Nat. Genet.
4:98-101; Ohlsson et al. (1993) Nature Genet. 4:94-97; Ogawa et al.
(1993b) Nature 362:749-751).
[0097] Furthermore, as discussed above, some tumors undergo loss of
imprinting (LOI) in cancer, with one or more of the following:
biallelic expression of IGF2 (Rainier et al. (1993) Nature
362:747-749; Ogawa et al. (1993b) Nature 362:749-751), epigenetic
silencing of H19 (Steenman et al. (1994) Nature Genet. 7:433-439;
Moulton et al. (1994) Nature Genet. 7:440-447); and/or abnormal
expression of the paternal H19 allele (Rainier et al. (1993) Proc.
Natl. Acad Sci. USA 85:6384-6388), and this observation has been
extended to a wide variety of childhood and adult malignancies
(Rainier et al. (1993) Proc. Natl. Acad. Sci. USA 85:6384-6388;
Suzuki et al. (1994) Nat. Genet. 6:332-333). Normal imprinting can
be maintained in part by allele-specific, tissue-independent
methylation of H19, since LOI is associated with abnormal
methylation of the normally unmethylated maternal H19 allele
(Steenman et al. (1994) Nature Genet. 7:433-439; Moulton et al.
(1994) Nature Genet. 7:440-447). There are other imprint-specific
marks of methylation that show alterations with LOI of 11p15 genes,
including KvLQT1 and others.
[0098] However, the previous studies did not report any comparison
of the rate of occurrence of LOI in the tumors or the matched
normal tissue of a cancerous population to the rate of occurrence
of LOI in the same tissue of a non-cancerous population. Thus, any
findings of LOI in the matched normal tissue of the cancerous
population have been previously explained in terms of
tissue-specific normal LOI. Such explanations are consistent with
the clonal theory of cancer, which attributes tumor generation to a
spontaneous mutation leading uncontrolled growth of a particular
clone (Norell, E C The Clonal Evolution of Tumor Cell Population,
1976 Science 149:23-28) and teaches against being able to detect a
genetic marker of a predisposition to contracting cancer in normal
cells. Biallelic expression of LGF2 in colorectal cancer has also
been ascribed to a change in promoter usage rather than LOI (Issa
et al. (1996) Proc. Natl. Acad. Sci. USA 93:11757-11762).
[0099] Moreover, the degree of LOI thus-far reported remains
unquantified and unquantifiable. There is no indication that there
is any relationship between the degree of LOI and the presence of
cancer or the likelihood of developing cancer.
[0100] Furthermore, the previous studies have only sought to detect
LOI in normal cells of the same tissue in which cancer is present.
There is no indication that there is any relationship between the
presence, absence, or degree of LOI in one tissue and the presence
or likelihood of developing cancer in a different tissue.
[0101] It would also be desirable to be able to make some
prediction or assessment about the chances of survival once a
patient has contracted cancer. It would be especially beneficial if
such predictions could be made before the patient actually
contracted cancer. With such a prediction in hand, it would be
possible to make better decisions about the examination of the
patient prior to the detection of cancer, as well as the treatment
of the patient after the detection of cancer.
[0102] Sudden infant death syndrome (SIDS) remains a significant
killer. It would be useful if infants could be screened for any
predisposition to SIDS, so preventative steps could be taken. SIDS
has recently been shown to be caused by heart arrhythmias caused by
prolongation of the long QT interval, which is controlled in part
by the KvLQT1 gene, which is normally imprinted. The imprinting of
KvLQT1 is disturbed in varying degrees in the infant population.
However, to date there is no method effective for screening infants
for the risk of SIDS.
[0103] The presence or absence of LOI may be detected by examining
any condition, state, or phenomenon which causes LOI or is the
result of LOI. Such conditions, states, and phenomena include, but
are not limited to
[0104] 1. Causes of LOI, such as the state or condition of the
cellular machinery for DNA methylation, the state of the imprinting
control region on chromosome 11, the presence of trans-acting
modifiers of imprinting, the degree or presence of histone
deacetylation;
[0105] 2. State of the genomic DNA associated with the genes or
gene for which LOI is being assessed, such as the degree of DNA
methylation;
[0106] 3. Effects of LOI, such as:
[0107] a. Relative transcription of the two alleles of the genes or
gene for which LOI is being assessed;
[0108] b. Post-transcriptional effects associated with the
differential expression of the two alleles of the genes or gene for
which LOI is being assessed;
[0109] c. Relative translational of the two alleles of the genes or
gene for which LOI is being assessed;
[0110] d. Post-translational effects associated with the
differential expression of the two alleles of the genes or gene for
which LOI is being assessed;
[0111] e. Other downstream effects of LOI, such as altered gene
expression measured at the RNA level, at the splicing level, or at
the protein level or post-translational level (i.e., measure one or
more of these properties of an imprinted gene's manifestation into
various macromolecules); changes in function that could involve,
for example, cell cycle, signal transduction, ion channels,
membrane potential, cell division, or others (i.e., measure the
biological consequences of a specific imprinted gene being normally
or not normally imprinted (for example, QT interval of the heart).
Another group of macromolecular changes could be in associated
processes such as histone acetylation, histone deacetylation, or
RNA splicing.
[0112] When detecting the presence or absence of LOI by relying on
any one of these conditions, states, or phenomena, it is possible
to use a number of different specific analytical techniques. In
particular, it is possible to use any of the methods for
determining the pattern of imprinting known in the art. It is
recognized that the methods may vary depending on the gene to be
analyzed.
[0113] Conditions, states, and phenomena which may cause LOI and
may be examined to assess the presence or absence of LOI include:
the state or condition of the cellular machinery for DNA
methylation, the state of the imprinting control region on
chromosome 11, the presence of trans-acting modifiers of
imprinting, the degree or presence of histone deacetylation or
histone deacetylation, imprinting control center, transacting
modulatory factors, changes in chromatin caused by polycomb-like
proteins, trithorax-like proteins, human homologues of other
chromatin-affecting proteins in other species such as Su(var)
proteins in Drosophila, SIR proteins in yeast, mating type
silencing in yeast, XIST-like genes in mammals.
[0114] It is also possible to detect LOI by examining the DNA
associated with the gene or genes for which the presence or absence
of LOI is being assessed. By the term "the DNA associated with the
gene or genes for which the presence or absence of LOI is being
assessed" it is meant the gene, the DNA near the gene, or the DNA
at some distance from the gene (as much as a megabase or more
away--i.e., methylation changes can be that far away, since they
act on chromatin over long distances). Such approaches include
measuring the degree of methylation in the DNA associated with the
gene or genes for which the presence or absence of LOI is being
assessed. It is also possible to detect LOI by examining
modifications to DNA-associated protein, such as histone
acetylation and histone deacetylation; changes to binding proteins
detected by band shift, protection assays, or other assays, in
addition to changes to the DNA sequence itself.
[0115] The degree of methylation in the DNA, associated with the
gene or genes for which the presence or absence of LOI is being
assessed, may be measured by means of a number of analytical
techniques. For example, the DNA, associated with the gene or genes
for which the presence or absence of LOI is being assessed, may be
sequenced using conventional DNA sequencing techniques as described
in Current Protocols in Molecular Biology. Asubel et al., Wiley
lnterscience, 1998, which is incorporated herein by reference. In
this case, the biological sample will be any which contains
sufficient DNA to permit sequencing.
[0116] In addition, the degree of methylation in the DNA,
associated with the gene or genes for which the presence or absence
of LOI is being assessed, may be measured by fluorescent in situ
hybridization (FISH) by means of probes which identify and
differentiate between genomic DNAs, associated with the gene for
which the presence or absence of LOI is being assessed, which
exhibit different degrees of DNA methylation. FISH is described in
the Human chromosomes: principles and techniques (Editors, Ram S.
Verma, Arvind Babu Verma, Ram S.) 2nd ed., New York: McGraw-Hill,
1995, and de Capoa A., Di Leandro M., Grappelli C., Menendez F.,
Poggesi I., Giancotti P., Marotta, M. R., Spano A., Rocchi M.,
Archidiacono N., Niveleau A. Computer-assisted analysis of
methylation status of individual interphase nuclei in human
cultured cells. Cytometry, 31:85-92, 1998 which is incorporated
herein by reference. In this case, the biological sample will
typically be any which contains sufficient whole cells or nuclei to
perform short term culture, Usually, the sample will be a tissue
sample which contains 10 to 10,000, preferably 100 to 10,000, whole
somatic cells.
[0117] Typically, in methods for assaying allele-specific gene
expression which rely upon the differential transcription of the
two alleles, RNA is reverse transcribed with reverse transcriptase,
and then PCR is performed with PCR primers that span a site within
an exon where that site is polymorphic (i.e., normally variable in
the population), and this analysis is performed on an individual
that is heterozygous (i.e., informative) for the polymorphism. One
then uses any of a number of detection schemes to determine whether
one or both alleles is expressed. See also, Rainier et al. (1993)
Nature 362:747-749; which teaches the assessment of allele-specific
expression of IGF2 and H19 by reverse transcribing RNA and
amplifying cDNA by PCR using new primers that permit a single round
rather than nested PCR; Matsuoka et al. (1996) Proc. Natl. Acad Sci
USA 93:3026-3030 which teaches the identification of a transcribed
polymorphism in p57.sup.KIP2; Thompson et al. (1996) Cancer
Research 56:5723-5727 which teaches determination of mRNA levels by
RPA and RT-PCR analysis of allele-specific expression of
p57.sup.KIP2; and Lee et al. (1997) Nature Genet. 15:181185 which
teaches RT-PCR SSCP analysis of two polymorphic sites. Such
disclosures are herein incorporated by reference. In this case, the
biological sample will be any which contains sufficient RNA to
permit amplification and subsequent reverse transcription followed
by polymerase chain reaction. Typically, the biological sample will
be a tissue sample which contains Ito 10,000,000, preferably 1000
to 10,000,000, more preferably 1,000,000 to 10,000,000, somatic
cells.
[0118] It is also possible to utilize allele specific
RNA-associated in situ hybridization (ASISH) to detect the presence
or absence of LOI by relying upon the differential transcription of
the two alleles. In ASISH, the relative abundance of transcribed
mRNA for two alleles is assessed by means of probes which identify
and differentiate between the mRNA transcribed from the two
alleles. Typically, the probes are tagged with fluorescent labels
which results in a high sensitivity and easily quantifiable
results. ASISH is described in Adam et al. (1996) "Allele-specific
in situ hybridization (ASISH) analysis: a novel technique which
resolves differential allelic usage of H19 within the same cell
lineage during human placental development," Development 122:83-47,
which is incorporated herein by reference. In this case, the
biological sample will typically be any which contains sufficient
whole cells or nuclei to perform histological section and in situ
hybridization. Usually, the sample will be a tissue sample which
contains 10-100,000, preferably 100-1000, whole somatic cells.
[0119] According to the present invention, it is also possible to
detect LOI by examining allele-specific post-transcriptional
effects (i.e., effects after transcription and before translation),
like alternate splicing that depends on which allele was
transcribed, and detection of secondary structure of the RNA.
[0120] It is also possible, according to the present invention, to
detect LOI by examining the relative translation of the two alleles
of the gene or genes for which the presence or absence of LOI is
being measured. In this case, the presence or relative abundance of
the two polypeptides arising from the expression of the two alleles
is measured directly. This approach can be effected by any known
technique for detecting or quantifying the presence of a
polypeptide in a biological sample. For example, allele-specific
translational effects may be examined by quantifying the proteins
expressed by the two alleles using antibodies specific for each
allele (transcribed, translated polymorphism). Such effects may be
measured and/or detected by such analytical techniques as Western
blotting, or use of an ELISA assay. In this case, the biological
sample will be any which contains a sufficient amount of the
polypeptide(s) encoded by the gene(s) for which the presence or
absence of LOI is being measured.
[0121] LOI may also be detected by examining post-translational
effects, such as secondary modifications that are specific to one
allele, like glycosylation or phosphorylation. For example, one
allele may be modified, say by phosphorylation or glycosylation,
and the other one not. Because the polymorphism encodes a
recognition motif, then one can readily distinguish the difference
by a Western blot, detecting alternate migration of the polypeptide
or protein; use of antibodies specific for the modified form;
radioactive incorporation of phosphoryl group or glycosyl group or
other modification (i.e., in living cells, followed by the
detection of a band at a varying location).
[0122] LOI may also be detected by reliance on other
allele-specific downstream effects. For example, depending on the
metabolic pathway in which lies the product of the imprinted gene;
the difference will be 2X versus 1X (or some number in between) of
the product, and therefore the function or a variation in function
specific to one of the alleles. For example, for IGF2, increased
mitogenic signaling at the IGFI receptor, increased occupancy of
the IGF1 receptor increased activity at the IGF2 catabolic
receptor, decreased apoptosis due to the dose of IGF2; for KvLQT1,
change in the length of the QT interval depending on the amount and
isoform of protein, or change in electrical potential, or change in
activity when the RNA is extracted and introduced into Xenopus
oocytes.
[0123] It is also possible to detect LOI by detecting an associated
halotype, i.e., linked polymorphisms that identify people whose
genes are prone to LOI.
[0124] In the examples described below, LOI is detected by relying
on a polymorphism, i.e., a genetic difference between the two
alleles. However, it will be recognized that many of the techniques
described above may be used to detect LOI even when there is no
polymorphism in the two alleles of the gene or genes for which the
presence or absence of LOI is being measured. For example, LOI may
be detected by reliance on allele-specific DNA methylation
(polymorphism independent); histone acetylation; other
modifications to DNA; or alterations in replication timing, when
the imprinted allele shows "replication timing asynchrony" i.e. the
two alleles replicate at different times. When the two alleles
replicate at the same time, LOI may be detected by FISH. Since
imprinted alleles pair in the late S phase, LOI may be detected by
the absence of such pairing in the late S as observed by FISH.
[0125] On the other hand certain techniques are more conveniently
used when there is a polymorphism in the two alleles of the gene or
genes for which the presence or absence of LOI is being measured.
For example, RT-PCR followed by SSCP (single strand conformational
polymorphism) analysis; restriction enzyme digestion analysis
followed by electrophoresis or Southern hybridization; or
radioisotopic PCR; PCR; allele-specific oligonucleotide
hybridization; direct sequencing manually or with an automated
sequencer; denaturing gradient gel electrophoresis (DGGE); and many
other analytical techniques can be used to detect LOI when relying
on a polymorphism.
[0126] The presence or absence of LOI may be determined for any
gene or genes which are known to normally exhibit imprinting.
Currently there are about 22 genes which are known to be normally
imprinted (see Feinberg in The Genetic Basis of Human Cancer, B
Vogelstein & K Kinzler, Eds., McGraw Hill, 1997, which is
incorporated herein by reference). Examples of such genes include,
but are not limited to, IGF2, H19, p57.sup.KIP2, KvLQT1, TSSC3,
TSSCS, and ASCL2. However, it is expected that additional genes
which normally exhibit imprinting will be discovered in the future
and the LOI of such genes may be the target of the present methods
and are therefore included in the present invention.
[0127] Direct approaches to identifying novel imprinted genes
include, but are not limited to, positional cloning efforts aimed
at identifying imprinted genes near other known imprinted genes
(Barlow et al. (1991) Nature 349:84-87); techniques comparing gene
expression in parthenogenetic embryos to that of normal embryos
(Kuroiwa et al. (1996) Nat. Genet. 12:186-190) and restriction
landmark genome scanning (Nagai et al. (1995) Biochem. Biophys.
Res. Commun. 213:258-265).
[0128] In a preferred embodiment, the gene or genes for which LOI
is detected is selected on the basis of which particular type of
cancer is sought to be detected. For example, if the screening test
is for the presence of colorectal cancer or the risk of contracting
colorectal cancer, it is preferred that the gene for which the
degree of LOI is determined is the IGF2 gene. It is contemplated as
part of the present invention that other genes may be associated
with colorectal cancer or with other types of cancers. It is
further contemplated that the present invention includes using the
techniques and procedures described herein for the diagnosis,
detection or determining the predisposition for other cancers and
other diseases.
[0129] If the biological sample of the subject in question is found
to exhibit LOI, then that subject is identified as having an
increased probability of having cancer. In this case, it may be
preferred to carry out further diagnostic tests to probe for the
possibility of cancer being present in the subject. Examples of
such further diagnostic tests include, but are not limited to,
chest X-ray, colorectal examination, endoscopic examination, MRI,
CAT scanning, or other imaging such as gallium scanning, and barium
imaging.
[0130] The method of screening for the risk of contracting cancer
is carried out much the same as the above-described method for
detecting the presence of cancer or other disease with the
exception that the detection of LOI in the biological sample of a
subject results in the subject as being classified as having an
increased risk of contracting cancer or other disease. In this
embodiment of the present invention, it may be preferred to perform
one or more of the above-described further diagnostic tests to
probe for the possibility of cancer being present in the subject.
In addition, it may also be preferred to prescribe a schedule for
performing additional diagnostic tests in the future, even if no
cancer is present at the time LOI is detected. For example, if LOI
is detected in a biological sample of a subject and indicates an
increased risk of contracting cancer, it may be preferred to
schedule periodic (e.g., every 1 to 12 months) chest X-rays,
colorectal examinations, endoscopic examination, MRI, CAT scanning,
other imaging such as gallium scanning, and/or barium imaging for
that subject.
[0131] Similarly, according to the present invention, if the
biological sample of the subject in question is found to exhibit
LOI, then that subject is identified as having an increased
probability of having replication repair error defects. In this
embodiment of the present invention, it may be preferred to carry
out the same further steps as described above in the context of
detecting cancer and detecting an increased risk of contracting
cancer.
[0132] In another preferred embodiment of the present invention
testing is performed by measuring the degree of LOI for a
particular gene or set of genes. By "the degree of LOI" it is meant
the percentage of total expression (as measured by actual
expression or transcription) attributable to the allele which is
normally imprinted. The degree of LOI may be calculated by allele
ratio, i.e., the more abundant allele divided by the less abundant
allele. In another embodiment, the degree of LOI may be calculated
by the following formula: 1 Degree of LOI = E i E i + E n in which
E i is the level of expression due to the allele which is norm
[0133] ally imprinted and E.sub.n is the level of expression due to
the allele which is normally expressed. Thus, the degree of LOI may
be determined by any method which allows the determination of the
relative expressions of the two alleles. For example, a degree of
LOI of 100% reflects complete LOI (equal expression of both
alleles), while a degree of LOI of 0% reflects no LOI (expression
of only one allele). Any method of measuring the relative
expression of the two alleles is considered to be included in the
present invention.
[0134] In a particularly preferred embodiment of the present
invention, the degree of LOI is measured for the IGF2 gene when
screening for the presence of colorectal cancer. In another
particularly preferred embodiment of the present invention, the
degree of LOI is measured for the IGF2 gene when screening for the
presence of stomach cancer. In another particularly preferred
embodiment of the present invention, the degree of LOI is measured
for the IGF2 gene when screening for the presence of esophageal
cancer. In another particularly preferred embodiment of the present
invention, the degree of LOI is measured for the IGF2 gene when
screening for the presence of leukemia.
[0135] According to the present invention, a number of the
techniques described above in the context of detecting LOI may also
be used effectively to measure the degree of LOI. For example, the
degree of LOI may be easily quantified by means of such techniques
as, e.g.:
[0136] 1. Measuring the alterations in DNA directly including, but
not limited to, methylation, histone acetylation, etc. by, for
example, FISH;
[0137] 2. Measuring the degree of relative transcription by ASISH;
RNA in situ hybridization to metaphase chromosomes;
[0138] 3. Measuring the degree of relative transcription by RT-PCR
of mRNA followed by a variety of detection schemes;
[0139] 4. Measuring the degree of relative translation by Western
blotting, ELISA, etc.; and
[0140] 5. DNA in situ hybridization to metaphase or interphase
chromosomes when change in replication timing, allele pairing, or
secondary structure can be detected;
[0141] When measuring the degree of LOI quantitatively by relying
on the relative levels of transcription of the two alleles, using
quantitative PCR amplification, it is important to obtain high
quality RNA. In this case, it is preferred to place the tissue in
an appropriate buffer and take those precautions known to and used
by those of ordinary skill in the art when isolating RNA. Such
techniques are described in Current Protocols in Molecular Biology,
Asubel et al., Wiley Interscience, 1998, which is incorporated
herein by reference in its entirety.
[0142] When assessing the degree of LOI quantitatively by a method
which relies on the relative transcription of the two alleles,
using quantitative PCR amplification, it is also important to avoid
genomic DNA contamination of the cDNA, which is obtained from the
mRNA. In addition, it is important to ensure linear amplification
during any amplification step, e.g., polymerase chain reaction
(PCR) amplification of the cDNA obtained from the mRNA. If the
primers used in such a PCR amplification are exhausted, it is
possible to obtain heterodimers of two different alleles, and any
subsequent restriction enzyme digestion will not reflect the true
ratio of expression of the two alleles. For example, if alleles are
a and b, and the corresponding cDNAs are a' and b', and the
restriction enzyme being used recognizes allele b, then the
restriction enzyme cuts the bb' double helix. However, if the PCR
amplification is allowed to progress to the point where the primers
are exhausted, it is possible to obtain after the final annealing
and extension a mixture of aa', ab', ba', and bb' rather than the
desired mixture of aa' and bb'. since alleles a and b may differ by
only a small number of nucleotide residues. The restriction enzyme
may then cut only bb' without cutting ab' and ba' resulting in a
false apparent 3:1 ratio of expression.
[0143] It is also preferred to use a linear detection platform. In
this regard, good results have been achieved by using a
PhosphorImager (model 445SI, manufactured by Molecular Dynamics)
which detects radioactive emissions directly from a gel. Other
linear detection systems include carefully titrated autoradiography
followed by image analysis, beta-emission detection analysis
(Betascan). Another linear detection platform is an automated DNA
sequencer such as ABI 377 analyzer.
[0144] It is important to note that, although in the Examples
provided below the presence of a polymorphism in the gene of
interest formed the basis for measuring the degree of LOI, as in
the case of detecting LOI, it is also possible to assess the degree
of LOI in a particular gene even when no polymorphism is present in
that gene. For example, imprinting can be assessed by the degree of
methylation of CpG islands in or near an imprinted gene (e.g.,
Barletta, Cancer Research, op. cit. In addition, imprinting can be
assessed by changes in DNA replication timing asynchrony, e.g.
White L M. Rogan P K. Nicholls R D. Wu B L. Korf B. Knoll J H.
Allele-specific replication of 15q11-q13 loci: a diagnostic test
for detection of uniparental disomy. American Journal of Human
Genetics. 59:423-30, 1996.
[0145] On the other hand, certain techniques are more conveniently
used when there is a polymorphism in the two alleles of the gene or
genes for which the presence or absence of LOI is being measured.
For example, RT-PCR, followed by gel electrophoresis to distinguish
length polymorphisms, or RT-PCR followed by restriction enzyme
digestion, or by automated DNA sequencing, or by single strand
conformational polymorphism (SSCP) analysis, or denaturing gradient
gel electrophoresis, etc.; or, completely DNA based methods that
exploit, for example DNA methylation (then there is no RT step, to
convert RNA to cDNA prior to PCR);
[0146] Once the degree of LOI has been measured for the gene or
genes in question, the risk of having cancer is then assessed by
comparing the degree of LOI for that gene or genes is to a known
relationship between the degree of LOI and the probability of the
presence of the particular type of cancer or other disease. The
relationship between the degree of LOI and the probability of the
presence of a particular type of cancer may be determined for any
combination of a normally imprinted gene or genes and a particular
type of cancer by determining
[0147] The method of screening for the risk of contracting cancer
is carried out much the same as the above-described method for
detecting the presence of cancer with the exception that the
measured degree of LOI is compared to a known relationship between
the degree of LOI and the probability of contracting the particular
type of cancer. The relationship between the degree of LOI and the
probability of contracting a particular type of cancer may be
determined by one of ordinary skill in the art for any combination
of a normally imprinted gene or genes and a particular type of
cancer by determining the degree of LOI in a statistically
meaningful number of tissue samples obtained from patients with
cancer, and determining the degree of LOI in a statistically
meaningful number of tissue samples obtained from patients without
cancer, and then calculating an odds ratio as a function of the
degree of LOI.
[0148] It should also be understood that the present methods of
detecting cancer, assessing the risk of contracting cancer, and
assessing the risk of having replication error repair defects may
be carried out by comparing the degree of LOI against one or more
predetermined threshold values, such that, if the degree of LOI is
below a given threshold value then the subject is assigned to a low
risk population for having cancer, contracting cancer, and/or
having replication error repair defects. Alternatively, the
analytical technique may be designed not to yield an explicit
numerical value for the degree of LOI, but instead yield only a
first type of signal when the degree of LOI is below a threshold
value and/or a second type of signal when the degree of LOI is
below a threshold value. It is also possible to carry out the
present methods by means of a test in which the degree of LOI is
signaled by means of a non-numeric spectrum such as a range of
colors encountered with litmus paper.
[0149] The present methods of detecting the presence of a disease,
assessing the risk of contracting a disease, and detecting the risk
of having replication may suitably be carried out on any subject
selected from the population as a whole. However, it may be
preferred to carry out this method on certain selected groups of
the general population when screening for the presence of
particular types of cancer. Preferably, the present method is used
to screen selected groups which are already known to have an
increased risk of contracting the particular type of cancer in
question.
[0150] It is to be understood that the present invention can be
performed on the general population to assess the presence or risk
of disease. In another embodiment of the present invention, target
patients may be tested to detect a particular type of disease, for
example colon cancer. In addition, according to the present
invention, subgroups of those patients who already are thought to
be at some increased risk, such as e.g., a weak family history, may
be tested.
[0151] In another embodiment, the present invention includes kits
which are useful for the detection of cancer and/or assessing the
risk of contracting cancer. According to the present invention, the
kits contain those components, ingredients, and/or means for
carrying out the present methods. The components contained in the
kit depend on a number of factors, including: the condition, state,
or phenomenon relied on to detect LOI or measure the degree of LOI,
the particular analytical technique used to detect LOI or measure
the degree of LOI, and the gene or genes for which LOI is being
detected or the degree of LOI is being measured.
[0152] In the embodiment of the present invention wherein LOI is
detected by relying on the degree of methylation of the genomic DNA
associated with the gene(s) for which LOI is being detected or the
degree of LOI is being measured using FISH, the kit will typically
contain one or more probes which can identify a specific imprinted
gene or group of genes. Typically, such probes will be nucleic
acids or monoclonal antibodies and will be linked to, for example,
a fluorescent label.
[0153] In the case of detecting LOI by relying on the differential
rates of transcription of two ploymorphic alleles, the kit may
comprise:
[0154] (i) means for the amplification of the mRNAs corresponding
to the two polymorphic alleles of the gene in question. Examples of
such means include suitable DNA primers for the PCR amplification
of the mRNAs corresponding to the two polymorphic alleles of the
gene in question. Specific examples of such means includes any pair
of DNA primers which will anneal to and amplify any gene which is
normally imprinted and in which a polymorphism is present.
[0155] According to the present invention, the kit may further
comprise:
[0156] (ii) means for identifying the products of the amplification
of the mRNAs corresponding to the two polymorphic alleles of the
gene in question.
[0157] Such means include, but is not limited to, a restriction
enzyme which specifically cleaves one of the products of the
amplification of the mRNAs corresponding to the two polymorphic
alleles of the gene in question. Specific examples of such enzymes
include, but are not limited to, Apa I in the case of the IGF2
gene.
[0158] In the embodiment of the present invention wherein the
degree of LOI is measured by relying on the differential rates of
transcription of two ploymorphic alleles, the kit may comprise:
[0159] (i) means for the linear amplification of the mRNAs
corresponding to the two polymorphic alleles of the gene in
question. Examples of such means include a sufficient quantity of
suitable DNA primers for the PCR amplification of the mRNAs
corresponding to the two polymorphic alleles of the gene in
question, such that the PCR amplification may be carried out
without exhausting the primers and linear amplification achieved.
Specific examples of such means includes any pair primers for any
gene which is normally imprinted.
[0160] According to the present invention, the kit may further
comprise:
[0161] (ii) means for identifying the products of the amplification
of the mRNAs corresponding to the two polymorphic alleles of the
gene in question. Such means include a restriction enzyme which
specifically cleaves one of the products of the amplification of
the mRNAs corresponding to the two polymorphic alleles of the gene
in question.
[0162] When detecting LOI or measuring the degree of LOI by ASISH,
the kit will typically contain one or more probes which can
identify and distinguish between the RNA associated with the two
alleles. Typically, such probes will be nucleic acids that are
specific for each allele, and are used either sequentially or
together using different fluorescent labels for each allele.
[0163] When detecting LOI or measuring the degree of LOI by
assessing the relative translation of two alleles, the kit may
contain antibodies that distinguish the protein product of the two
alleles.
[0164] In another embodiment of the present invention, a method for
screening infants/newborns is provided for the risk of SIDS. In
this embodiment, the imprinting pattern of a tissue sample of a
newborn is examined. Detection of an abnormal imprinting pattern in
the tissue sample indicates that the infant/newborn is at risk from
SIDS. The tissue sample of the infant/newborn is preferably blood,
and the detection of the imprinting pattern in the tissue may be
determined for any of the genes discussed above. It preferred that
the imprinting pattern be determined for KvLQT1. KvLQT1 is normally
imprinted in all tissues except the heart. Detection of an abnormal
imprinting pattern for KvLQT1 will result in that infant/newborn
being categorized as being at risk from SIDS. When an
infant/newborn is so categorized, it may be preferred to carry out
further steps such as prescribing nocturnal monitoring, etc.
[0165] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLE 1
[0166] Material
[0167] Eighty specimens derived from colorectal cancer patients
were analyzed for the presence of heterozygosity of either of
either an Apa I or CA repeat polymorphism in exon 9 of the human
IGF2 gene (Rainier et al. (1993) Nature 362:747-749) that can be
used to assess allele-specific expression by reverse transcription
polymerase chain reaction (RT-PCR), of which 27 were heterozygous
and thus informative for imprinting status analysis. Sixteen
informative of 47 normal colon samples were used as controls.
Fifteen informative of 40 peripheral blood samples were further
examined for IGF2 imprinting status. There were no significant
differences between the and noncancer patients for age, race, or
sex. (FIG. 1)
[0168] Nucleic Acid Preparation
[0169] DNA and RNA were extracted from snap-frozen tissues and
peripheral blood leukocytes. For DNA, tissues or cells were
incubated at 55.degree. C. overnight in a lysis buffer containing
1X TE, 0.5% SDS, 150 mM NaCl and 100 .mu.g/ml proteinase K,
followed by extraction with phenol/chloroform and ethanol
precipitation. RNA was extracted with RNAzoI B (Tel-test) following
the manufacturer's instructions.
[0170] Quantitative Analysis of IGF2 Imprinting Status
[0171] As described in the Results, many samples demonstrated
substantial although incomplete loss of imprinting. A quantitative
PCR assay (relative to the two alleles) was developed (also see
FIG. 1). RNA samples were treated with DNase prior to making any
cDNA, in order to avoid any possible genomic DNA contamination. The
reaction was performed in a 25 .mu.l volume, using 10 .mu.l RNA
(5-10) .mu.g), 5 X transcription buffer (Promega), 0.7 .mu.l RNasin
(Promega), and 2 .mu.l RNase-free DNase I (Boehringer-Mannheim), at
25.degree. C. for 30 mm. The RNA was extracted with phenol and
chloroform sequentially, and then ethanol-precipitated together
with 20 pmol of RT primer P10 in exon 9. cDNA was generated in a 25
.mu.l reaction containing 5 .mu.l X RT buffer (LTI), 2.5 .mu.l dNTP
(10 mM), 0.7 .mu.l RNasin (Promega), 2.5 .mu.l sodium pyrophosphate
(40 mM), and 2 .mu.l AMV reverse transcriptase (LTI), at 42.degree.
C. for 1 hour. As a control, all reactions were performed in
duplicate in the presence and absence of reverse transcriptase.
Either the Apa I or (CA).about.repeat polymorphism was used to
analyze imprinting status of the gene. To exclude any possibility
of genomic DNA contamination, PCR across an intron-exon boundary
was first performed. For the Apa I polymorphism, primer Pla,
located on exon 8, and primer P8b, on exon 9, were used to amplify
cDNAs derived from reverse transcription. PCR was performed using
the following conditions: SO pl of reaction volume containing 2 pl
of cDNA template, at a final concentration of 0.5 .mu.M each
primer, 0.15 mM of dNTP, 1.5 mM of MgCl.sub.2, 1 X PCR buffer
(LTI), and 1.5 U of Taq polymerase (LTI). Thermal cycling was
performed as follows: 94.degree. C. for 2 minutes; 32 cycles at
94.degree. C. for 1 minute, 52.degree. C. for 1 minute, 72.degree.
C. for 1.5 minutes, and 72.degree. C. for 10 minutes. The
PCR-amplified products were purified from 1.5% agarose gels, using
a 123 bp ladder to identify the location of the cDNA (1224 bp, as
distinguished from genomic DNA at 1513 bp). This additional step
made any genomic DNA contamination impossible. The cDNA fragments
were purified using the Qiaquick gel extraction kit (Qiagen). A
second round of PCR amplification was then performed using 8 .mu.l
of purified first round PCR product as template, and primers P2 and
P3, with P3 previously labeled using [Y.sup.32P1-ATP. The PCR
product (16 .mu.l) was digested in a 20 .mu.l volume with 20 U Apa
1, 10 mM NaCl, 3 mM MgCl.sub.2, pH 7.5, and electrophoresed on a 6%
denatured polyacrylamide gel. The (CA). repeat region was analyzed
using primers P8 and labeled P9, and visualized directly on a 6%
denatured polyacrylamide gel. Each allele was then quantified on a
PhorphorImager (Molecular Dynamics), as percent of the less
abundant allele (0% representing monoallelic expression, 100%
representing equal biallelic expression). Primers were maintained
in excess over PCR product to avoid heterodimer formation. Control
mixing experiments confirmed equal amplification of the two
alleles, and the absence of heterodimer formation in the Apa I
assay. Samples were analyzed in duplicate with an assay to assay
variation of 0-10%, and the results for each sample were averaged.
Statistical analysis among the various groups revealed significant
quantitative differences, as described in the Results. This
analysis allowed us to define a threshold level for partial LOI as
50% (i.e., <3:1 ratio of the more abundant to less abundant
allele). Primer sequences were as follows:
1 P1a, 5'-ATCGTTGAGGAGTGCTGTTTC-3'; P2,
5'-CTTGGACTTTGAGTCAAATTGG-3; P3, 5'-GGTCGTGCCAATTACATTTCA-3'; P8,
5'-CTCATACTTTATGCATCCCCG-3'; P8b, 5'-CGGGGATGCATAAAGTATGAG-3'; P9,
5'-GCCTGATCCATACAGATATCG-3'; P10, 5'-GCATCTCTGTCATGGTGGAAAG-3'.
[0172] Promoter-Specific Allele Usage Analysis
[0173] IGF2 specific cDNA was made as described above. A
semi-nested PCR approach was performed using promoter-specific
primers to amplify transcripts derived from specific promoters as
described previously (He and Cui, 1998). Duplicate PCR products
were separated on 1.5% agarose gels, and the DNA fragments
migrating at the predicted specific cDNA size were isolated and
purified. Southern bLOI hybridization was then performed using
allele-specific oligonucleotide probes that discriminated the Apa I
within exon 9 of IGF2. The conditions for Southern allele-specific
hybridization (SASH) were as follows: oligonucleotide probes were
prepared by 3' end-labeling with .sup.32P-dATP and purification as
described previously (Cui 1997). The filter was hybridized at
45.degree. C. overnight, followed by stringent washing using 0.1
SSC/0.1% SDS at 55.degree. C., for 10 minutes for oligonucleotide
probe A (without the Apa I site) and 5 minutes for probe B (with
Apa I site). A reconstitution experiment was done in parallel to
ensure accurate quantitation. Oligonucleotide probe sequences were:
probe A, 5-TGTGATTTCTGGGGTCCTTCTTTTCTCTT-3, probe B,
5'-TGTGATTTCTGGGGCCCTTCTTT- TCTCTT-3.
[0174] DNA Microsatellite Instability Analysis
[0175] DNA microsatellite instability is assessed by comparing
tumor and matched normal genomic DNA, using the following 15
microsatellite markers for each sample: BAT-25, BAT-26, D2S123,
D11S1318, D17S250, AP2, D10S89, AP3, D18S58, D3S1283, D11S904,
D11S1758, D11S4124, D11S860, and APC. PCR amplification was
performed using 1 .mu.l of DNA (.apprxeq.0.15 .mu.g) in a final
volume of 10 .mu.l, with a final concentration of 0.1 .mu.M each
primer, 0.15 mM dNTP, 1.5 mM MgCl.sub.2, 1 X PCR buffer (LTI), and
0.06 U Taq polymerase. In each case, one primer was end-labeled.
PCR products were analyzed on 6% denatured polyacrylamide gels. The
primer sequences were as described previously (Dietmaier et al.
(1997) Cancer Res. 57:4749-4756) (Genome Database, Johns Hopkins
University, http://gdb.www.gcb.org).
[0176] Statistical Analysis
[0177] Cross-tabulation and comparison of sample means were used to
identify differences between colon cancers and normal mucosa with
and without LOI, between samples with and without microsatellite
instability, and to examine differences in the characteristics of
cancer patients with and without LOI. Where appropriate, the
chi-square test of independence and student's t-test were applied
to the data to determine significant differences. Quantitative LOI
was compared between cancers and matched normal mucosa using a
paired sample t-test.
EXAMPLE 2
[0178] LOI in Colorectal Cancers and Matched Normal Mucosal
Samples
[0179] Of the 27 tumors informative for IGF2, 12 (44%) showed
substantial expression of the less abundant allele (LOI was defined
as <3:1 ratio of the more abundant to less abundant allele).
Surprisingly, in all 10 cases in which the tumor showed LOI and
matched normal colonic mucosa was available from the same patient,
the matched normal tissue also exhibited LOI (FIG. 2a; patients 1,
2, 4). In contrast, only 1 of 12 (8.5%) matched normal mucosal
specimens showed LOI when the matched tumor did not show LOI (FIG.
2b; patients 19, 21). This difference was highly statistically
significant (p<0.001).
[0180] To avoid any arbitrariness in scoring LOI, the quantitative
level of LOI of each tumor was compared to that of the matched
normal mucosa of each patient. The degree of LOI in the tumor
correlated strongly with the degree of LOI in matched normal tissue
(FIG. 3; r.sup.2=0.726, p<0.001). A paired student's t-test also
showed no significant difference between the degree of LOI in the
paired tumor and normal specimens. LOI in matched normal mucosa was
not due to contaminating tumor cells, because in every case, the
matched normal tissue was derived from the colon >10 cm from the
tumor, and the normal specimens were all verified to be free of
tumor, dysplasia, or any other histopathological abnormality. Thus,
LOI was a property of the colon itself in most patients with LOI,
affecting both the normal mucosa and the cancer.
EXAMPLE 3
[0181] No Change in Promoter Usage in Tumors with LOI
[0182] The IGF2 gene contains four promoters, P1-P4, and only P2-P4
are normally imprinted. To confirm that biallelic expression was
due to LOI and not to a shift in promoter usage to P1,
promoter-specific RT-PCR was performed, using exon-specific primers
(exon 3 for P1, exon 4 for P2, exon 5 for P3, and exon 6 for P4).
The PCR products were then analyzed using allele-specific
oligonucleotides as described (He et al. (1998) Oncogene
16:113-119), with reconstitution controls performed in parallel. In
every case tested, biallelic expression in both tumor and normal
specimens was observed from P3 and P4, which are both normally
imprinted, and not from P1 (FIG. 4). Thus, biallelic expression in
both tumor and matched normal specimens reflected abnormal
imprinting, rather than a shift in promoter usage.
[0183] This result is in contrast to that of Issa et al., who
reported expression from the adult P1 promoter in colon cancer, and
not from the imprinted promoters P2-P4 (Issa et al. (1996) Proc.
Natl. Acad. Sci. USA 93:11757-11762). However, that group examined
cell lines rather than primary tumors, and it has been shown
directly that there is promoter-specific LOI from P3 and P4 in
primary colon cancers examined directly.
EXAMPLE 4
[0184] LOI Linked to Microsatellite Instability
[0185] Of the 27 informative cancers, 10 showed replication errors
of at least two of 15 microsatellite markers tested. Tumors showing
microsatellite instability showed a mean expression of the less
abundant allele of 39.5%, compared to 16.9% for the cancers without
microsatellite instability, a statistically significant difference
(p<0.001). These results were essentially the same if tumors
with only one microsatellite instability were included, as there
was only one such additional tumor. Similarly, matched normal
tissues corresponding to tumors with microsatellite instability
showed a mean expression of the less abundant allele of 40.4%,
compared to 13.2% for the matched normal tissues corresponding to
the tumors without microsatellite instability (p<0.001). When
the two groups were compared with regard to <3:1 ratio of more
abundant allele to less abundant allele (the recommended threshold
for substantial LOI). the results were again statistically
significant. Of the 10 cases with microsatellite instability of at
least two markers, all 10 exhibited substantial LOI (100%; Table
1). In contrast, only 2 of 16 (12.5%) tumors without microsatellite
instability showed LOI above this threshold (p<0.001; Table 1).
Thus, LOI was specifically associated with microsatellite
instability in the tumors. Similarly, all 9 (100%) of the matched
normal mucosal specimens corresponding to tumors with two or more
microsatellite instability showed LOI, compared to 14% (2/14)
matched normal mucosal specimens corresponding to tumors without
microsatellite instability (p<0.001; Table 1).
[0186] The presence of LOI was also significantly associated with
the quantitative number of microsatellite instability among the 15
markers tested. The mean number of microsatellite instability among
tumors with LOI was 4.7.+-.1.2, compared to 0.07.+-.0.07 for the
tumors without LOI (p<0.001). Thus, both subtle and substantial
defects in replication error repair were linked to LOI in the
colon.
EXAMPLE 5
[0187] LOI in Patients Without Known Cancer
[0188] These data suggest that LOI in the normal colonic mucosa
identifies a subset of patients who show LOI in their cancers, and
that this subgroup frequently exhibits MSI in their tumors. If that
is the case, then the frequency of LOI in a population of patients
without cancer should be similar to the frequency of LOI in the
matched normal mucosa of patients who did not show LOI in their
tumors. To test this hypothesis, 47 normal colon mucosal specimens
were obtained from patients who did not have cancer, of which 16
were informative for IGF2. While many of these specimens showed
very low levels of expression of the less abundant allele, only 2
(12.5%, Table 1) showed a <5:1 ratio of allele-specific
expression (both <2:1), the same proportion seen in the matched
normal mucosa of patients whose tumors did not show MSI (FIG.
4).
[0189] In addition, the presence of LOI in the normal colon of
colon cancer patients with LOI in their tumors suggests that this
abnormality may be present ubiquitously in at least some of these
patients. To test this hypothesis, four blood specimens were
obtained from patients with LOI in their tumors and normal colon,
and all four showed LOI (FIG. 4, FIG. 5). As a control, 40 blood
specimens were obtained from a general hospital chemistry
laboratory, of which 15 were informative for IGF2, and only 2 of
these unselected specimens exhibited substantial or complete LOI
(13.3%, p<0.01), similar to the proportion seen in the normal
mucosa of noncancer patients. These data suggest that, at least in
some patients, abnormal imprinting may be detected in the blood of
cancer patients. The results of these Examples is summarized in
Table I
2 TABLE I LOI in Normal Colon No LOI in Normal Colon Matched
Matched Cancer Normal Cancer Normal P % Ex- 39.5 .+-. 7.0 40.4 .+-.
6.1 16.9 .+-. 14.1 13.3 .+-. 11.9 <0.001 pression of less
abundant allele % With 91 NA 11 NA <0.001 replication errors
Mean # 4.7 NA 0.07 NA <0.001 markers with replication errors Age
48.7 .+-. 5.5 62.9 .+-. 4.5 <0.01 Sex 5 M, 6 F 8 M, 8 F N.S.
Race N.S. % in stage 45 NA 50 NA N.S. 3-4 % at 44 NA 50 NA 0.6
proximal location
[0190] Summary
[0191] According to the present invention, it has been determined
that frequent loss of imprinting (LOI) in colon cancer, and in the
matched normal colonic mucosa of the same patients, as well as in
blood samples of 4 patients. This study has two major implications.
First, it represents the first genetic abnormality detected at high
frequency in the normal tissue of cancer patients in the general
population. It is not known whether LOI develops concurrently with
the cancer or precedes the development of the cancer. This question
will require prospective studies both before and after cancer
diagnosis. However, the fact that cancer patients with LOI in their
normal mucosa developed cancer on average 14 years younger than
those without LOI, suggests that LOI precedes the cancer.
[0192] Second, these results demonstrate directly a link between
abnormal genomic imprinting and DNA replication errors, because LOI
in both normal and cancer tissue was linked both qualitatively and
quantitatively to MSI in the tumors. Imprinted chromosomal regions
show asynchronous replication between the two parental chromosomes
over a relatively large (several megabase) region (Kitsbert et al.
(1993) Nature 364:459-463). Furthermore, the two parental
homologues are physically associated in late S phase LaSalle, J M
and Lalande, M. (1996) Science 272:725-728. LOI does not
necessarily detect mutations in conventional HNPCC genes that cause
instability in a high proportion of microsatellite markers, as not
all tumor cell lines from HNPCC patients show LOI. Rather, it is
hypothesized that the altered imprinting described here and the
common non-HNPCC related MSI with which it is associated, are due
to disturbances in chromatin that affect both replication and
imprinting fidelity.
[0193] These results help to resolve a paradox in studies of MSI in
colorectal cancer. The frequency of MSI in sporadically occurring
tumors is 15-37%, approximately half with MSI-H and half with
MSI-L. However, germline mutations in genes known to cause MSI are
seen in <2% of colon cancer patients, and in only 16% of
sporadic tumors with MSI-H and almost none with MSI-L. Thus it has
been unclear whether most of the patients with these tumors develop
mutations in DNA repair genes in somatic cells during the
progression of the tumor, or whether most of these errors are due
to as yet unknown germline mutations. The data presented here
suggest that MSI precedes the development of cancer. This has an
important practical implication, as the traditional assays for MSI
require a clonal cell population (a tumor) to compare to a normal
cell population, whereas the assay of LOI does not.
[0194] Slightly more than 10% of patients in the general
population, not known to have cancer, also showed LOI in normal
colonic mucosa and in blood. These patients may have cancer or at
substantially increased risk of cancer, since LOI was specifically
associated with MSI in colorectal cancer. MSI in many common
tumors, including those of the stomach, colon, and lung, is
associated with a younger age, positive family history, and/or less
accessible and detectable location, suggesting that a relatively
large subgroup of cancer patients in the general population are at
increased risk of cancer and show MSI in their tumors, even though
they do not fall within a well-defined syndrome. Some of the
patients with LOI in normal tissue are therefore also be at risk of
cancers other than colorectal cancer, since MSI-L may be more
strongly associated with familial and/or younger onset lung and
stomach cancer than with colorectal cancer.
[0195] Finally, the assay of genomic imprinting described here is
of considerable practical importance, as this assay does not
require tumor tissue. The approach described here also represents
the first genetic test that ascertains a substantial fraction of
patients in the general population with cancer or at risk of
cancer.
[0196] It should be understood that the foregoing relates only to a
preferred embodiment of the present invention and that numerous
modifications or alterations may be made therein without departing
from the spirit and the scope of the invention as set forth in the
appended claims.
* * * * *
References