U.S. patent application number 13/204737 was filed with the patent office on 2011-12-01 for methods and kits for determining a risk to develop cancer, for evaluating an effectiveness and dosage of cancer therapy and for correlating between an activity of a dna repair enzyme and a cancer.
This patent application is currently assigned to YEDA RESEARCH AND DEVELOPMENT CO., LTD.. Invention is credited to SARA BLUMENSTEIN, ZVI LIVNEH, TAMAR PAZ-ELIZUR.
Application Number | 20110294134 13/204737 |
Document ID | / |
Family ID | 26973412 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110294134 |
Kind Code |
A1 |
LIVNEH; ZVI ; et
al. |
December 1, 2011 |
METHODS AND KITS FOR DETERMINING A RISK TO DEVELOP CANCER, FOR
EVALUATING AN EFFECTIVENESS AND DOSAGE OF CANCER THERAPY AND FOR
CORRELATING BETWEEN AN ACTIVITY OF A DNA REPAIR ENZYME AND A
CANCER
Abstract
Methods and kits for (i) determining a risk of a subject to
develop cancer; (ii) evaluating an effectiveness and dosage of
cancer therapy administered to a cancer patient; and (iii)
determining a presence of correlation or non-correlation between an
activity of at least one DNA repair enzyme and at least one cancer,
are disclosed.
Inventors: |
LIVNEH; ZVI; (REHOVOT,
IL) ; PAZ-ELIZUR; TAMAR; (REHOVOT, IL) ;
BLUMENSTEIN; SARA; (RAMAT GAN, IL) |
Assignee: |
YEDA RESEARCH AND DEVELOPMENT CO.,
LTD.
REHOVOT
IL
|
Family ID: |
26973412 |
Appl. No.: |
13/204737 |
Filed: |
August 8, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10469992 |
Sep 16, 2003 |
|
|
|
PCT/IL02/00231 |
Mar 21, 2002 |
|
|
|
13204737 |
|
|
|
|
09815015 |
Mar 23, 2001 |
|
|
|
10469992 |
|
|
|
|
60303338 |
Jul 9, 2001 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/19 |
Current CPC
Class: |
C12Q 2600/158 20130101;
G01N 33/574 20130101; G01N 2333/924 20130101; G01N 2333/988
20130101; C12Q 1/6886 20130101; C12Q 2600/106 20130101; A61P 35/00
20180101; G01N 2800/52 20130101 |
Class at
Publication: |
435/6.14 ;
435/19 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/44 20060101 C12Q001/44 |
Claims
1. A method of determining a risk of a subject to develop cancer,
the method comprising determining a level of catalytic activity of
a DNA repair enzyme in a biological sample of the subject, wherein
said DNA repair enzyme is selected from the group consisting of
8-oxoguanine DNA glycosylase (OGG1), AP endonuclease1 (APE1),
methylpurine DNA glycosylase (MPG), uracil DNA glycosylase1 (UNG1),
uracil DNA glycosylase2 (UNG2), SMUG1, MBD4, mismatch-specific
thymine/uracil glycosylase (TDG), enodonuclease III (NTH1),
adenine-specific mismatch DNA glycosylase (MYH),
8-oxo-GTPase/8-oxodGTPase (MTH1), dUTPase (DUT), AP endonuclease2
(APE2), deoxyribose phosphate lyase (POLB) and wherein a level of
said activity below a predetermined value is indicative of an
increased risk of the subject to develop said cancer.
2. The method of claim 1, wherein said cancer is selected from the
group consisting of lung cancer, colorectal cancer and head and
neck cancer.
3. The method of claim 1, wherein the subject is known to be, or is
about to be, exposed to environmental conditions associated with
increased risk of developing said cancer.
4. The method of claim 3, wherein said environmental conditions are
selected from the group consisting of smoking and occupational
exposure to smoke or ionizing radiation.
5. The method of claim 1, wherein when said level of catalytic
activity of said subject is below said predetermined level,
indicative of increased risk of said subject to develop cancer,
further counseling said subject to avoid exposure to ionizing
radiation or smoke.
6. The method of claim 1, wherein said DNA-repair enzyme is APE1
and said cancer is lung cancer or colorectal cancer.
7. The method of claim 6, wherein when said level of catalytic
activity of said subject is below said predetermined level,
indicative of increased risk of said subject to develop cancer,
further counseling said subject to avoid exposure to ionizing
radiation or smoke.
8. The method of claim 1, wherein said DNA-repair enzyme is MPG and
said cancer is lung cancer or colorectal cancer.
9. The method of claim 8, wherein when said level of catalytic
activity of said subject is below said predetermined level,
indicative of increased risk of said subject to develop cancer,
further counseling said subject to avoid exposure to ionizing
radiation or smoke.
10. A method of determining a risk of a subject to develop
colorectal cancer, the method comprising determining a level of
catalytic activity of 8-oxoguanine DNA glycosylase in a sample of
peripheral blood lymphocytes of the subject and, according to said
level, determining the risk of the subject to develop colorectal
cancer, wherein a level of said activity below a predetermined
value is indicative of an increased risk of said subject to develop
colorectal cancer.
11. The method of claim 10, wherein said level of catalytic
activity is determined using a double-stranded DNA substrate
comprising the complementary oligonucleotides having a
polynucleotide sequence as set forth in SEQ ID NOs: 1 and 2.
12. The method of claim 10, wherein when said level of catalytic
activity of said subject is below said predetermined level,
indicative of increased risk of said subject to develop cancer,
further counseling said subject to avoid exposure to ionizing
radiation or smoke.
13. A method of determining a risk of a subject to develop head and
neck cancer, the method comprising determining a level of catalytic
activity of 8-oxoguanine DNA glycosylase in a sample of peripheral
blood lymphocytes of the subject and, according to said level,
determining the risk of the subject to develop head and neck
cancer, wherein a level of said activity below a predetermined
value is indicative of an increased risk of said subject to develop
head and neck cancer.
14. The method of claim 13, wherein said level of catalytic
activity is determined using a double-stranded DNA substrate
comprising the complementary oligonucleotides having a
polynucleotide sequence as set forth in SEQ ID NOs: 1 and 2.
15. The method of claim 13, wherein when said level of catalytic
activity of said subject is below said predetermined level,
indicative of increased risk of said subject to develop cancer,
further counseling said subject to avoid exposure to ionizing
radiation or smoke.
16. A method of predicting the efficacy of a mutagenic cancer
treatment in a subject, the method comprising determining a level
of catalytic activity of a DNA repair enzyme in a biological sample
of the subject, and, according to said level, predicting the
efficacy of the mutagenic anti-cancer treatment in the subject,
wherein a level of said activity below a predetermined value is
indicative of an increased efficacy of said mutagenic cancer
treatment, wherein said DNA repair enzyme is selected from the
group consisting of 8-oxoguanine DNA glycosylase (OGG1), AP
endonuclease1 (APE1), methylpurine DNA glycosylase (MPG), uracil
DNA glycosylase1 (UNG1), uracil DNA glycosylase2 (UNG2), SMUG1,
MBD4, mismatch-specific thymine/uracil glycosylase (TDG),
enodonuclease III (NTH1), adenine-specific mismatch DNA glycosylase
(MYH), 8-oxo-GTPase/8-oxodGTPase (MTH1), dUTPase (DUT), AP
endonuclease2 (APE2), deoxyribose phosphate lyase (POLB).
17. The method of claim 16, wherein said mutagenic anti-cancer
treatment is selected from the group of chemotherapy and
radiotherapy.
18. The method of claim 16, further comprising selecting dosage of
said mutagenic anti-cancer treatment for treating the subject.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/469,992 filed on Sep. 16, 2003, which is a National
Phase of PCT Patent Application No. PCT/IL02/00231 filed on Mar.
21, 2002, which is a Continuation-In-Part (CIP) of U.S. patent
application Ser. No. 09/815,015 filed on Mar. 23, 2001, now
abandoned, and which also claims the benefit of priority of U.S.
Provisional Patent Application No. 60/303,338 filed on Jul. 9,
2001.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to the field of diagnosis and
prognosis. More particularly, the present invention relates to
methods of and kits for (i) determining a risk of a subject to
develop cancer; (ii) evaluating an effectiveness and preferred
dosage of cancer therapy administered to a cancer patient; and
(iii) determining a presence of correlation or non-correlation
between an activity of at least one DNA repair enzyme and at least
one cancer.
[0003] The DNA in each cell of a body is constantly subjected to
damage caused by both internal (e.g., reactive oxygen species) and
external DNA damaging agents (e.g., sunlight, X- and .gamma.-rays,
smoke) (Friedberg, et al., 1995). Most lesions are eliminated from
DNA by one of several pathways of DNA repair (Friedberg, et al.,
1995, Hanawalt, 1994, Modrich, 1994, Sancar, 1994). When unrepaired
DNA lesions are replicated, they cause mutations because of their
miscoding nature (Echols and Goodman, 1991, Livneh, et al., 1993,
Strauss, 1985). The occurrence of such mutations in critical genes,
e.g., oncogenes and tumor suppressor genes, may lead to the
development of cancer (Bishop, 1995, Vogelstein and Kinzler, 1993,
Weinberg, 1989). Indeed, DNA repair has emerged in recent years as
a critical factor in cancer pathogenesis, as a growing number of
cancer predisposition syndromes have been shown to be caused by
mutations in genes involved in DNA repair and the regulation of
genome stability. These include Xeroderma Pigmentosum (Weeda, et
al., 1993), Hereditary nonpolyposis colon cancer (Fishel, et al.,
1993, Leach, et al., 1993, Modrich, 1994, Parsons, et al., 1993),
Ataxia Telangiectasia (Savitsky, et al., 1995), Li-Fraumeni
syndrome (Srivastava, et al., 1990), and the BRCA1 (Gowen, et al.,
1998, Scully, et al., 1997) and BRCA2 genes (Connor, et al., 1997,
Patel, et al., 1998, Sharan, et al., 1997). In these cases, which
represent a minority of the cancer cases, gene mutations have
caused malfunction, leading to a strong reduction in DNA
repair.
[0004] A possible extension of the role of DNA repair in hereditary
cancer, would be a role for DNA repair in sporadic cancer. Several
studies suggested that inter-individual variability in DNA repair
correlates with variation in cancer susceptibility, with low repair
correlated to higher cancer risk (Athas, et al., 1991, Helzlsouer,
et al., 1996, Jyothish, et al., 1998, Parshad, et al., 1996, Patel,
et al., 1997, Sagher, et al., 1988, Wei, et al., 1996, Wei, et al.,
1993, Wei, et al., 1994).
[0005] 7,8-dihydro-8-oxoguanine (also termed 8-oxoguanine or
8-hydroxyguanine; dubbed 8-OxoG) is formed in DNA by two major
pathways: (a) Modification of guanine in DNA by reactive oxygen
species formed by intracellular metabolism, oxidative stress,
cigarette smoke, or by radiation (Asami, et al., 1997, Gajewski, et
al., 1990, Hutchinson, 1985, Leanderson and Tagesson, 1992). (b)
Incorporation into DNA by DNA polymerases of 8-oxo-dGTP, which is
formed by oxidation of intracellular dGTP (Maki and Sekiguchi,
1992). Once in DNA, 8-oxoG is replicated by DNA polymerases with
the misinsertion of dAMP, causing characteristic GC to TA
transversions (Shibutani, et al., 1991, Wood, et al., 1990). When
the modified dGTP is used as a substrate by DNA polymerases, it is
often misinserted opposite an A in the template, causing AT to CG
transversions (Pavlov, et al., 1994).
[0006] The major route for removing 8-oxoG from DNA is base
excision repair, initiated by 8-oxoguanine DNA N-glycosylase,
product of the OGG1 gene (in humans termed also hOGG1; (Aburatani,
et al., 1997, Arai, et al., 1997, Bjoras, et al., 1997, Radicella,
et al., 1997, Roldan-Arjona, et al., 1997, Rosenquist, et al.,
1997). The OGG1 gene was recently knocked-out in mice, such that
the effects on carcinogenesis can now be examined in this organism
(Klungland, et al., 1999, Minowa, et al., 2000). Expression of the
E. coli enzyme in Chinese hamster cells reduced 4-fold the
mutagenicity of .gamma. radiation (Laval, 1994), indicating that
the repair of 8-oxoG is important in negating the mutagenic
activity of .gamma. radiation. The following observations associate
OGG1 with cancer: (i) OGG1 was mapped to chromosome 3p25, a site
frequently lost in human lung and kidney cancers (Arai, et al.,
1997, Audebert, et al., 2000, Ishida, et al., 1999, Lu, et al.,
1997, Wikman, et al., 2000). (ii) OGG1 was found to be mutated in 2
out of 25 lung tumors (Chevillard, et al., 1998), and in 4 out of
99 renal tumors (Audebert, et al., 2000). (iii) OGG1 was found to
be mutated in a leukemic cell line (Hyun, et al., 2000) and in a
gastric cell line (Shinmura, et al., 1998). (iv) Analysis of p53
mutations in human lung, breast, and kidney tumors revealed a
substantial occurrence of GC to TA mutations, a mutation type
produced by unrepaired 8-oxoG (Hollstein et al., 1996;
Hernandez-Boussard, et al., 1999).
[0007] Since preventive measures which reduce the risk of
developing cancer, such as, but not limited to, the use of
anti-oxidants, diet, avoiding cigarette smoking, refraining from
occupational exposure to cancer causing agents, are known and
further since periodic testing and therefore early detection of
cancer offers improved cure rates, there is a great need for, and
it would be highly advantageous to have methods and kits for
determining a risk of a subject to develop cancer.
[0008] Since the effectiveness of cancer therapy depends on the
sensitivity of cells to genotoxic (mutageic) agents, there is a
great need for, and it would be highly advantageous to have methods
and kits for evaluating an effectiveness and preferred dosage of
cancer therapy administered to a cancer patient.
[0009] There is also a great need for, and it would be highly
advantageous to have methods and kits for determining a presence of
correlation or non-correlation between an activity of at least one
DNA repair enzyme and at least one cancer, so as to allow to
determine a risk of a subject to develop cancer and to evaluate an
effectiveness and preferred dosage of cancer therapy administered
to a cancer patient.
SUMMARY OF THE INVENTION
[0010] According to one aspect of the present invention there is
provided method of determining a risk (e.g., odds ratio, relative
risk) of a subject to develop cancer, the method comprising
determining a level of a parameter indicative of a level of
activity of a DNA repair/damage preventing enzyme in a tissue of
the subject, and, according to the level, determining the risk of
the subject to develop the cancer.
[0011] According to another aspect of the present invention there
is provided a method of determining a risk of a subject to develop
cancer, the method comprising determining (a) a presence or absence
of exposure to environmental conditions, such as smoking and
occupational exposure to smoke or ionizing radiation, associated
with increased risk of developing cancer; and (b) a level of a
parameter indicative of a level of activity of a DNA repair/damage
preventing enzyme in a tissue of the subject; and according to the
presence or absence and the level, determining the risk of the
subject to develop the cancer.
[0012] According to still another aspect of the present invention
there is provided a method of determining a presence of correlation
or non-correlation between an activity of at least one DNA
repair/damage preventing enzyme and at least one cancer, the method
comprising determining a level of a parameter indicative of a level
of activity of at least one DNA repair/damage preventing enzyme in
tissue derived from a plurality of cancer patients and a plurality
of apparently normal individuals, and, according to the level
determining the correlation or non-correlation between the activity
of the at least one DNA repair/damage preventing enzyme and the at
least one cancer.
[0013] According to further features in preferred embodiments of
the invention described below, the parameter is selected from the
group consisting of a protein level of said DNA repair/damage
preventing enzyme, a level of a RNA encoding said DNA repair/damage
preventing enzyme and a level of catalytic activity of said DNA
repair/damage preventing enzyme.
[0014] According to still further features in the described
preferred embodiments the cancer is selected from the group
consisting of lung cancer, blood cancers, colorectal cancer, breast
cancer, prostate cancer, ovary cancer and head and neck cancer.
[0015] According to still further features in the described
preferred embodiments the tissue is selected from the group
consisting of blood cells, scraped cells and biopsies.
[0016] According to still further features in the described
preferred embodiments the DNA repair/damage preventing enzyme is
selected from the group consisting of a DNA N-glycosylase,
deoxyribose phosphate lyase and AP endonuclease.
[0017] According to still further features in the described
preferred embodiments the DNA N-glycosylase is selected from the
group consisting of Uracil DNA glycosylase, hSMUG1, hMBD4,
Mismatch-specific thymine/uracil glycosylase, Methylpurine DNA
glycosylase, hNTH1, Adenine-specific mismatch DNA glycosylase and
8-oxoguanine DNA glycosylase.
[0018] According to still further features in the described
preferred embodiments the risk is expressed as a fold risk increase
as is compared to a normal, apparently healthy, population, or a
reference control group.
[0019] According to still further features in the described
preferred embodiments the risk is expressed in enzyme specific
activity units.
[0020] According to still further features in the described
preferred embodiments the risk is expressed as a magnitude of a
scale.
[0021] According to still further features in the described
preferred embodiments determining the level of catalytic activity
of the DNA repair/damage preventing enzyme is effected using a DNA
substrate having at least one lesion therein.
[0022] According to still further features in the described
preferred embodiments the at least one lesion is at a predetermined
site in the DNA substrate.
[0023] According to still further features in the described
preferred embodiments the lesion is selected from the group
consisting of uracil, 5-fluorouracil, 5-hydroxyuracil, isodialuric
acid, alloxan, uracil or thymine in U/TpG:5meCpG, uracil (U:G),
3,N.sup.4-ethenocytosine, (eC:G), T (T:G), 3-methyladenine,
7-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine, 1,
N6-ethenoadenine, 1,N2-ethenoguanine, thymine glycol, cytosine
glycol, dihydrouracil, formamidopyrimidine urea, adenine from A:G;
A:8-oxoG; C:A, 2-hydroxyadenine, 2,5-amino-5-formamidopyrimidine,
7,8-dihydro-8-oxoguanine (also termed 8-oxoguanine) and abasic
site.
[0024] According to still further features in the described
preferred embodiments the substrate includes at least two different
lesions of at least two types.
[0025] According to still further features in the described
preferred embodiments the substrate includes a single lesion.
[0026] According to still further features in the described
preferred embodiments the substrate includes at least two different
lesions of a single type.
[0027] According to still further features in the described
preferred embodiments the subject is known to be, or is about to
be, exposed to environmental conditions associated with increased
risk of developing cancer.
[0028] According to yet another aspect of the present invention
there is provided a method of predicting the efficacy of a
mutagenic anti-cancer treatment, such as chemotherapy and/or
radiotherapy, in a subject, the method comprising determining a
level of a factor indicative of a level of activity of a DNA
repair/damage preventing enzyme in a tissue of the subject, and,
according to the level, predicting the efficacy of the mutagenic
anti-cancer treatment in the subject.
[0029] According to still another aspect of the present invention
there is provided a method of selecting dosage of a mutagenic
anti-cancer treatment, such as chemotherapy and/or radiotherapy,
for treating a subject, the method comprising determining a level
of a factor indicative of a level of activity of a DNA
repair/damage preventing enzyme in a tissue of the subject, and,
according to the level, selecting dosage of the mutagenic
anti-cancer treatment for treating the subject.
[0030] According to an additional aspect of the present invention
there is provided a kit for determining a level of activity of a
DNA repair/damage preventing enzyme in a tissue of a subject, the
kit comprising, a package including, contained in sealable
containers, a DNA substrate having at least one lesion therein and
a reaction buffer.
[0031] According to further features in preferred embodiments of
the invention described below, the kit, further comprising test
tubes for separating lymphocytes.
[0032] According to still further features in the described
preferred embodiments the test tubes are prepackaged with an
anti-coagulant.
[0033] According to still further features in the described
preferred embodiments the kit further comprising a liquid having a
specific gravity selected effective in separating lymphocytes from
red blood cells via centrifugation.
[0034] According to still further features in the described
preferred embodiments the kit further comprising a solution having
osmolarity selected effective in lysing red blood cells.
[0035] According to still further features in the described
preferred embodiments the kit further comprising a protein
extraction buffer.
[0036] According to still further features in the described
preferred embodiments the kit further comprising reagents for
conducting protein determinations.
[0037] According to still further features in the described
preferred embodiments the kit further comprising a purified DNA
repair/damage preventing enzyme, which serves as a control for such
activity.
[0038] The present invention successfully addresses the
shortcomings of the presently known configurations by providing,
methods, kits and reagents useful in determining a risk of a
subject to develop cancer and for evaluating an effectiveness and
individual dosage of cancer therapy administered to a cancer
patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0040] In the drawings:
[0041] FIG. 1a shows an outline of an OGGA nicking assay according
to the present invention. In the assay a 32 base pair synthetic DNA
is cleaved at an 8-oxoG lesion (indicated by a circle), generating,
after denaturation, a radiolabeled 17-mer. The asterisk represents
a radiolabeled phosphate group.
[0042] FIGS. 1b-c represent a time course of the OGGA nicking assay
of the present invention, performed under standard conditions, with
a protein extract prepared from peripheral blood lymphocytes from a
healthy donor. FIG. 1b shows a phosphorimage of the reaction
products fractionated by urea-PAGE, and FIG. 1c shows the
quantification of the images. GO, the DNA substrate with a
site-specific 8-oxoG; G, a control substrate with a G instead of
8-oxoG.
[0043] FIGS. 2a-b show protein titration in the OGGA nicking assay.
The assay was performed under standard conditions, with the
indicated amounts of protein extract prepared from peripheral blood
lymphocytes from a healthy donor. FIG. 2a shows a phosphorimage of
the reaction products fractionated by urea-PAGE, and FIG. 2b shows
the quantification of the images. GO, the DNA substrate with a
site-specific 8-oxoG; G, a control substrate with a G instead of
8-oxoG.
[0044] FIGS. 3a-b show analysis of the specificity of the OGGA
nicking assay of the present invention. The assay was performed
under standard conditions, except that the reaction mixture
contained 2 pmol of radiolabeled substrate containing 8-oxoG, and
the indicated amounts of unlabeled competing DNA. FIG. 3a shows a
phosphorimage of the reaction products fractionated by urea-PAGE,
and FIG. 3b shows the quantification of the images. The protein
extract was from a healthy donor Hx, G and GO represent unlabeled
competing DNAs, which were similar to the radiolabeled substrate,
and contained either hypoxanthine, guanine or 8-oxoG in the same
location.
[0045] FIG. 4 shows the OGGA distribution in healthy individuals
(i.e., control subjects). The OGGA nicking assay of the present
invention was performed with blood samples from 123 healthy donors.
OGGA.ltoreq.5.5 is defined as Low (less than 4% of the control
group) OGGA.gtoreq.5.5 is defined as Normal.
[0046] FIG. 5 shows a comparison of OGGA in males and females. The
OGGA distribution of the 123 individuals shown in FIG. 4, was
plotted separately for males (N=53) and females (N=70).
[0047] FIG. 6 shows a comparison of OGGA in smokers and
non-smokers. The OGGA distribution of the 123 individuals shown in
FIG. 4, was plotted separately for smokers (N=35) and non-smokers
(N=88).
[0048] FIG. 7 shows a comparison of OGGA in two age groups. The
OGGA distribution of the 123 individuals shown in FIG. 4, was
plotted separately for ages<50 (N=34) and .gtoreq.50 (N=89)
[0049] FIGS. 8a-d show OGGA in apparently healthy individuals and
in patients with breast cancer or chronic lymphocytic leukemia
(CLL). FIG. 8a--OGGA distribution of a control group of 70 healthy
female individuals (see FIG. 5), and of 31 breast cancer patients
(FIG. 8b). FIG. 8c--OGGA distribution in the control group of 123
subjects, and 19 CLL patients (FIG. 8d).
[0050] FIGS. 9a-b show OGGA in apparently healthy individuals and
in patients with lung cancer (NSCLC). FIG. 9a--OGGA distribution of
the control group of 123 healthy individuals (see FIG. 4), and of
102 lung cancer (NSCLC) patients (FIG. 9b).
[0051] FIGS. 10a-b show OGGA in apparently healthy individuals and
in lymphoma patients. FIG. 10a--OGGA distribution of the control
group of 123 healthy individuals (see FIG. 4), and of 18 lymphoma
patients (FIG. 10b).
[0052] FIGS. 11a-b show OGGA in apparently healthy individuals and
in patients with colorectal cancer. FIG. 11a--OGGA distribution of
the control group of 123 healthy individuals (see FIG. 4), and of
16 colorectal cancer patients (FIG. 11b).
[0053] FIGS. 12a-b are schematic representations of monomolecular
(FIG. 12a) and plurimolecular (FIG. 12b) universal substrates in
accordance with the teachings of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] The present invention is of methods and kits which can be
used for (i) determining a risk (e.g., odds ratio, relative risk)
of a subject to develop cancer; (ii) evaluating an effectiveness
and dosage of cancer therapy administered to a cancer patient; and
(iii) determining a presence of correlation or non-correlation
between an activity of at least one DNA repair/damage preventing
enzyme and at least one cancer.
[0055] The principles and operation of a method and kit according
to the present invention may be better understood with reference to
the drawings and accompanying descriptions.
[0056] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0057] While conceiving the present invention it was hypothesized
that inter-individual variations in DNA repair/damage preventing
activity modulate susceptibility of developing cancer.
[0058] While reducing the present invention to practice an
experimental system which is easily adaptable to clinical use was
developed, such that a defined DNA repair activity can now be used
in determining cancer risk, and be utilized as a tool in cancer
prevention, early detection and prognosis. Since the repertoire of
DNA lesions is very large, at present experimental focus was given
to an abundant and mutagenic DNA lesion, 8-oxoguanine (also termed
7,8-dihydro 8-oxoguanine or 8-hydroxyguanine; dubbed 8-oxoG).
However, other mutagenic DNA lesions, such as, but not limited to,
those listed in Table 3 below, can be similarly used to implement
the methods of the invention, following suitable adaptation.
[0059] Thus, while reducing the present invention to practice,
whether inter-individual variations in the activity of OGG,
correlate with increased susceptibility to several types of cancers
was studied. A lower repair activity might lead to an increased
load of DNA lesions, and therefore to increased mutation rate, and
earlier occurrence of cancer. Similarly, a lower repair activity
renders cancer cells more susceptible to cancer therapy, which is
genotoxic by nature. It should be noted that different types of DNA
repair may be critical in different types of cancer. The present
invention is exemplified, in a non-limiting fashion, with respect
to the removal from DNA of a specific type of mutagenic lesion,
8-oxoG, by the activities of one or more DNA N-glycosylase repair
enzymes, present in protein extracts from peripheral blood
lymphocytes.
[0060] The present invention is herein exemplified with respect to
the use of the level of the DNA repair enzymatic activity of DNA
N-glycosylase(s) directed toward 7,8 dihydroxy 8-oxoguanine
(8-oxoguanine DNA N-glycosylase activity; OGG), as a risk factor
for lung cancer, lymphomas, and colorectal cancer. The enzymatic
activity is measured in a protein extract extracted from peripheral
blood lymphocytes and is referred to herein interchangeably as the
OGGA nicking assay, OGGA assay or OGGA test.
[0061] Using the OGGA test, a case-control study was conducted on
309 individuals: 123 healthy individuals, and a total of 186 cancer
patients as follows: 102 lung cancer (NSCLC) patients, 31 breast
cancer patients, 18 lymphoma patients, 19 CLL patients, and 16
colorectal cancer patients. The following results were found.
[0062] The mean OGGA in healthy individuals of ages<50
(7.6.+-.0.9; N=34) was slightly higher than in healthy individuals
of ages.gtoreq.50 (7.0.+-.1.0; N=89). The difference is
statistically significant (P=0.02).
[0063] The mean OGGA in healthy men (7.3.+-.1.0; N=53) was similar
to healthy women (7.1.+-.1.0; N=70), the difference was not
statistically significant (P=0.36).
[0064] The mean OGGA in smokers (7.3.+-.1.00; N=35) was similar to
that of non-smokers (7.1.+-.1.0; N=88; P=0.46), indicating that the
smoking status had a negligible effect on OGGA.
[0065] The mean OGGA in lung cancer patients (6.0.+-.1.5; N=102)
was significantly lower than in healthy individuals (7.2.+-.1.0;
N=123), with P=0.0001.
[0066] A strong association was found between low OGGA and lung
cancer with odds ratio varying from 3.9 (95% CI 1.7-8.6, P=0.0009),
to 9.0 (95% CI 3.2-25.0, P=0.0001), depending on the definition of
the cutoff level (.ltoreq.7.3 and .ltoreq.5.6, respectively). This
indicates that a low OGGA value is a risk factor in lung
cancer.
[0067] The mean OGGA in lymphoma patients (6.2.+-.1.8; N=18) was
significantly lower than in healthy individuals (7.2.+-.1.0;
N=123), with P=0.0001.
[0068] Low repair is defined as OGGA value.ltoreq.5.5 units/.mu.g
protein, representing <4% of the healthy individuals. Normal
repair is defined as OGGA>5.5 units/.mu.g protein. After
adjustment for age, lymphoma patients were 15 times more likely
than the healthy controls to have a low OGGA value (Odds Ratio
15.2; 95% confidence interval, 3.7-62.5). This provides evidence
that a low OGGA value is a risk factor in lymphoma.
[0069] The data shows that OGGA was low in 2 out of 16 (12%)
colorectal cancer patients (compared to 5/123 i.e., 4.1% among
healthy individuals), indicating that low OGGA is a risk factor in
colorectal cancer.
[0070] OGGA distribution was normal in breast cancer patients,
indicating that OGGA is not a risk factor in this type of
cancer.
[0071] It will be appreciated that the OGGA and similar tests for
other DNA repair activities can be used for screening individuals
for purposes of prevention, early diagnosis and prognosis of
cancers. These uses will be described in more detail below.
[0072] The following provides examples:
[0073] (i) Screening for smokers who have low OGGA in order to
prevent lung cancer.
[0074] Although 85% of lung cancer patients are smokers, the great
majority of smokers deal well with carcinogenic effects of smoking,
and does not develop lung cancer. Even among heavy smokers,
approximately 90% do not develop the disease (Mattson et al, 1987;
Minna et al, 2002). The results presented herein clarifies the fact
that the combination of smoking and low OGGA causes a dramatic
increase in susceptibility to lung cancer. For example, the
estimated risk of 30-years old smokers, with an OGGA value of 3.0,
is 221-fold higher than the reference (30-years old non-smokers
with an OGGA value of 7.0). For comparison, the estimated risk of
30-years old non-smokers, with an OGGA value of 3.0 is only 12-fold
higher than the reference. The simplest explanation for this
finding is that smokers with Low OGGA in peripheral blood
lymphocytes have a lower OGGA also in their lungs. Having a low
repair to start with, smoking causes further overloading of DNA
damage, therefore leading to a high cancer risk. This is a
classical example in which the risk of developing cancer is a
combination of genetic factors (level of DNA repair) and external
factors (cigarette smoking). Such individuals may be persuaded to
quit smoking. Such a screen will be effective as a preventive means
against lung cancer, and will lead eventually to a decrease in the
incidence of lung cancer.
[0075] (ii) Avoiding occupational hazard.
[0076] A considerable amount of people work in places which deal
with radiation or with smoke. These include radiology departments
in hospitals, nuclear industry, nuclear reactors, army personal
dealing with nuclear weapons, etc. These people can be tested for
OGGA, as a mandatory test, for their own safety. Individuals with
Low OGGA might have an increased probability of developing cancer
in such places, since ionizing radiation and smoke each produce
8-oxoG. Such individuals will be advised to seek an alternative
working environment.
[0077] (iii) Using the OGGA value as a prognostic marker for cancer
therapy. Cancer therapy relies heavily on chemicals and radiation.
These agents act, in most cases, by inflicting massive DNA damage,
which leads to selective killing of the rapidly dividing cancer
cells. The problem with such therapeutic agents is that they
damage, or kill, also non-cancer cells. Knowing the level of OGGA
in a cancer patient, may be used as a marker to estimate the
prognosis of a particular therapeutic treatment.
[0078] (iv) Screening for susceptibility to lymphoma or colorectal
cancers.
[0079] OGGA can be used to screen individuals for susceptibility to
lymphomas or colorectal cancer.
[0080] (v) Early detection of cancer.
[0081] Individuals with low OGGA (e.g., smokers with low OGGA who
would not quit smoking) can be advised to undergo periodical
follow-ups, in order to enable early detection of lung cancer.
[0082] Thus, according to one aspect of the present invention there
is provided a method of determining a risk of a subject to develop
cancer. The method according to this aspect of the present
invention is effected by determining a level of a factor indicative
of a level of activity of a DNA repair/damage preventing enzyme in
a tissue of the subject, and, according to the level, determining
the risk of the subject to develop the cancer.
[0083] As used herein throughout the term "indicative of" includes
correlating to.
[0084] According to another aspect of the present invention there
is provided a method of determining a risk of a subject to develop
cancer. The method according to this aspect of the present
invention is effected by determining a presence or absence of
exposure to environmental conditions, such as smoking and
occupational exposure to smoke or ionizing radiation, associated
with increased risk of developing cancer; and determining a level
of a factor indicative of a level of activity of a DNA
repair/damage preventing enzyme in a tissue of the subject; and
according to the presence or absence and the level, determining the
risk of the subject to develop the cancer.
[0085] Anyone of several approaches may be exploited according to
the present invention in determining a level of a factor indicative
of a level of activity of a DNA repair/damage preventing enzyme in
a tissue of the subject.
[0086] According to one embodiment a protein level of the DNA
repair/damage preventing enzyme is determined, which is indicative
of the level of activity of the DNA repair/damage preventing
enzyme. Several alternative quantitative assays are available for
determining protein levels. Each of which is based on the specific
interactions between proteins and antibodies specific thereto.
Table 1 below lists known antibodies recognizing different DNA
repair/damage preventing enzyme.
TABLE-US-00001 TABLE 1 Enzyme Antibody Source/Reference 1. Uracil
DNA PU101 Slupphaug et al., 1995.sup.(1) glycosylase (UNG) 2.
8-oxoguanine AB1a331 Monden et al., 1999.sup.(2) glycosylase (OGG1)
Anti-human OGG1 Alexis Biochemicals 3. Adenine mismatch Anti-hMYH
.alpha. 344 Parker et al., 2000.sup.(3) glycosylase (MYH) Anti-hMYH
.alpha. 516 Parker et al., 2000.sup.(3) 4. 8-oxodGTPase Anti-M78
Kang et al., 1995.sup.(4) (MTH1) 5. dUTPase (DUT) DUT415 Ladner et
al., 1997.sup.(5) 6. AP endonuclease I Ref-1 (H-300), Ref-1 Santa
Cruz (HAP1, APE1, (C-20), Ref-1 (N-16), Biotechnology REF1, APEX)
Ref-1 (E-17) 7. Deoxyribose mAb-10S Srivastava et al., 1995.sup.(6)
phosphate lyase (of 18S mAb Srivastava et al., 1999.sup.(7) DNA
polymerase .beta.) .sup.(1)Slupphaug, G., Eftedal, I., Kavli, B.,
Bharati, S., Helle, N. M., Haug, T., Levine, D. W., Krokan, H. E.
(1995) Properties of a recombinant human uracil-DNA glycosylase
from the UNG gene and evidence that UNG encodes the major
uracil-DNA glycosylase. Biochemistry 34, 128-138. .sup.(2)Monden,
Y., Arai, T., Asano, M., Ohtsuka, E., Aburatani, H., Nishimura, S.
(1999) Human MMH (OGG1) type 1a protein is a major enzyme for
repair of 8-hydroxyguanine lesions in human cells. Biochem.
Biophys. Res. Comm. 258, 605-610. .sup.(3)Parker, A., Gu, Y. and
Lu, A. -L. (2000) Purification and characterization of a mammalian
homolog of Escherichia coli MutY mismatch repair protein from calf
liver mitochondria. Nucleic Acids Res. 28, 3206-3215. .sup.(4)Kang,
D., Nishida, J., Iyama, A., Nakabeppu, Y., Furuichi, M., Fujiwara,
T., Sekiguchi, M. and Takeshige, K. (1995) Intracellular
localization of 8-oxo-dGTPase in human cells, with special
reference to the role of the enzyme in mitochondria. J. Biol. Chem.
270, 14659-14665. .sup.(5)Ladner, R. D. and Caradonna, S. J. (1997)
The human dUTPase gene encodes both nuclear and mitochondrial
isoforms. J. Biol. Chem. 272 19072-19080. .sup.(6)Srivastava, D.
K., Rawson, T. Y., Showalter, S. D. and Wilson, S. H. (1995)
Phorbol ester abrogates up-regulation of DNA polymerase .beta. by
DNA-alkylating agents in Chinese hamster ovary cells. J. Biol.
Chem. 270, 16402-16408. .sup.(7)Srivastava, D. K., Husain, I.,
Arteaga, C. L. and Wilson, S. H. (1999) DNA polymerase .beta.
expression differences in selected human tumor cell lines.
Carcinogenesis 20, 1049-1054.
[0087] Antibodies recognizing any specific protein can be readily
elicited using methods well known in the art in which cells of an
immune system are exposed in vivo or in vitro to at least one
epitope of the protein of interest, preferably a plurality of
epitopes thereof. Such antibodies can be polyclonal or monoclonal.
Commercial antibody developing services are available throughout
the world. Examples include: Antibody Solutions, Palo Alto, Calif.,
Washington Biotechnology Inc., Baltimore, Md.; TNB Laboratories
Inc., St. John's, Newfoundland, Canada; and Genemed Synthesis Inc.,
South San Francisco, Calif.
[0088] Such antibodies can be used in a variety of well known
antibody based detection assays, including, but not limited to,
Western blot, ELISA, a protein chip assay and an antibody chip
assay.
[0089] In Western blot, total protein preparation is
electrophoresed typically under denaturing and optionally under
reducing conditions through a gel, typically a polyacrylamide gel.
Then, the proteins are blotted onto a membrane, which is thereafter
blocked by a non-specific protein, such as milk proteins. An
antibody specific to the protein of interest is then interacted
with the blot. The antibody will quantitatively bind to the protein
of interest. The binding between the antibody and the protein of
interest can be monitored by either directly labeling the antibody,
or, preferably using a labeled secondary antibody capable of
recognizing the first.
[0090] In ELISA the antibody capable of binding the protein of
interest is linked to an enzyme capable of catalyzing a
colorimetric reaction, which serves for quantitative detection.
[0091] In a protein chip assay, the protein of interest, typically
a plurality of different proteins of interest, are linked to a
solid support in addressable positions, so as to form a matrix of
proteins. An antibody or several antibodies specific to certain
proteins, each being labeled by a distinguishable label, are
interacted with the support in the presence of proteins derived
from a biological sample. A protein recognized by an antibody and
which is present in the biological sample will compete with its
solid support bound counterpart, such that the level of binding of
the antibody to the respective addressable location on the support,
is determinable by such competition for binding.
[0092] In an antibody chip assay, antibodies are linked to a solid
support in addressable positions, so as to form a matrix of
antibodies each capable of binding a different protein. Proteins
derived from a biological sample are labeled and the labeled
proteins are interacted with the solid support. The level of
binding to the solid support is determined, being indicative of the
level of the protein in the sample.
[0093] These assays are well known and are well described in the
art literature. Further details are available in, for example,
"Current Protocols in Immunology" Volumes I-III Coligan J. E., ed.
(1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th
Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and
Shiigi (eds), "Selected Methods in Cellular Immunology", W.H.
Freeman and Co., New York (1980); available immunoassays are
extensively described in the patent and scientific literature, see,
for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752;
3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074;
3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771
and 5,281,521; "Immobilized Cells and Enzymes" IRL Press, (1986);
"Methods in Enzymology" Vol. 1-317, Academic Press; Marshak et al.,
"Strategies for Protein Purification and Characterization--A
Laboratory Course Manual" CSHL Press (1996).
[0094] According to another embodiment of the present invention the
level of a RNA, such as mRNA, encoding the DNA repair/damage
preventing enzyme is determined, which is also indicative of the
level of activity of the DNA repair/damage preventing enzyme.
Several alternative quantitative assays are available for
determining RNA levels. Each of which is based on the specific
interactions between complementary nucleic acids. Table 2 below
lists the human genes encoding DNA repair/damage preventing
enzyme.
TABLE-US-00002 TABLE 2 Accession GI No. Enzyme Gene (SEQ ID NO:)
No. (NCBI) 1. Uracil DNA UNG (3) NM_003362 6224978 glycosylase 2.
SMUG1 SMUG1 (4) NM_014311 7657596 3. MBD4 MBD4 (5) NM_003925
4505120 4. Thymine TDG (6) NM_003211 4507422 glycosylase 5.
Methylpurine MPG (7) NM_002434 4505232 glycosylase 6. Endonuclease
III NTH1 (8) NM_002528 6224977 human homolog 7. Adenine mismatch
MYH (9) NM_012222 6912519 glycosylase 8. 8-oxoguanine OGG1 (10)
NM_002542 7949101 glycosylase 9. 8-oxodGTPase MTH1, NUDT1 (11)
NM_002452 4505274 10. dUTPase DUT (12) NM_001948 4503422 11. AP
endonuclease I APE1, HAP1. NM_001641 4502136 APEX, REF1 (13) 12.
Deoxyribose POLB (14) NM_002690 4505930 phosphate lyase (of DNA
polymerase .beta.)
[0095] Yet undescribed human genes of DNA repair/damage preventing
enzyme can nowadays be readily isolated using in-silico searches,
since the majority (nearly all) of the coding sequences of the
human genome have been cloned and sequenced. Traditional methods of
gene isolation can also be exploited as is further described in the
list of references provided hereinbelow.
[0096] Based on gene sequences, Northern blot, quantitative RNA PCR
(also known as quantitative RT-PCR), RNA dot blot and nucleic acid
chip assays can be readily developed and used to determine the
level of a specific RNA, such as mRNA, in a biological sample.
Further details concerning these assays are available in, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., eds. (1984); "PCR Protocols: A Guide To Methods
And Applications", Academic Press, San Diego, Calif. (1990).
[0097] In an alternative embodiment, and as is further described in
detail below and exemplified by the Examples section that follows,
the factor which is determined is the catalytic activity per se of
the DNA repair/damage preventing enzyme.
[0098] The present invention is useful in determining a risk of a
subject to develop cancer, whereby any type of cancer is subject to
risk determination by way of implementing the method of the
invention. It is well known that all cancers arise from DNA
mutations and that the progress of a specific cancer from a primary
tumor to a metastatic tumor, reflects clonal selection of cancer
cells that accumulate mutations as they develop and turn more
cancerous (e.g., proliferate more rapidly, escape proliferation
control, acquire autosignalling behavior, induce angiogenesis,
etc.) and more metastatic. This process is subject to variations
depending on the specific genes involved in the development and
progression of different cancers. It is therefore expected that
different in vivo DNA repair/damage preventing activities are
required to prevent the formation of different cancers. Also, the
level of exposure of body tissues to genotoxic agents such as smoke
and radiation, differs. Since different types of genotoxic agents
cause different types of DNA lesions, it is again expected that
different in vivo DNA repair/damage preventing activities are
required to prevent the formation of different cancers.
[0099] The results obtained while reducing the present invention to
practice are in agreement with the above, as low OGGA was found to
be associated with some, but not all cancers tested. However,
assays similar to the OGGA assay described herein can be readily
developed for correlating other cancers with one or more DNA
lesions, some of which are listed in Table 3 below.
[0100] In effect, all known cancers can be evaluated by finding
correlation or non-correlation between the occurrence thereof and
the occurrence of low DNA repair/damage preventing activity of
certain types. When positive correlation is identified, a
predictive risk determination assay can be readily implemented.
[0101] Thus, according to an aspect of the present invention there
is provided a method of determining a presence of correlation or
non-correlation between an activity of at least one DNA
repair/damage preventing enzyme and at least one cancer. The method
according to this aspect of the invention is effected by
determining a level of activity of at least one DNA repair/damage
preventing enzyme in tissue derived from a plurality of cancer
patients and a plurality of apparently normal individuals, and,
according to the level determining the correlation or
non-correlation between the activity of the at least one DNA
repair/damage preventing enzyme and the at least one cancer. This
aspect of the invention is exemplified herein with respect to a
single DNA repair enzyme activity (8-oxoguanine DNA glycosylase)
using a suitable substrate having a single lesion therein, for a
plurality of cancers, for some correlation was found, whereas for
other, non-correlation was found.
[0102] Thus, the methods of determining a risk of a subject to
develop cancer described herein can be implemented for a variety of
cancers, including, but not limited to, lung cancers, e.g.,
small-cells lung cancer and non-small cells lung cancer, blood
cancers, e.g., lymphomas and leukemias, including, for example,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic
leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia and the like, colorectal cancer,
breast cancer, prostate cancer, ovary cancer, malignant melanoma,
stomach cancer, pancreas cancer, urinary cancer; uterus cancer,
bone cancer, liver cancer, thyroid cancer, brain cancer; head and
neck cancer, including, for example, salivary carcinoma and
laryngeal carcinoma.
[0103] DNA repair/damage preventing activity can be measured in
extracts of different body tissues or cells, which may be collected
from a testee by known methods. Blood cells, scraped cells (e.g.,
mouth or skin scrapes) and biopsies are good examples as such
tissues are routinely removed from subjects for diagnostics.
[0104] Several types of DNA repair/damage preventing activities can
be assayed according to the present invention, e.g., DNA
N-glycosylase, nucleotide pool sanitizing activity (dNTPase
activity, e.g., 8-oxodGTPase), AP endonuclease and deoxyribose
phosphate lyase (of DNA polymerase .beta.).
[0105] An assay for determining the activity of a DNA N-glycosylase
is described and exemplified herein with respect to 8-oxoguanine
DNA glycosylase. In this respect it is convenient to monitor the
nicking activity of DNA N-glycosylase towards DNA substrates
including one or more lesion.
[0106] An assay for monitoring the activity of 8-oxodGTPase is, for
example, as described by Mo et al. [Mo, J.-Y., Maki, H. and
Sekiguchi, M. (1992) Proc. Natl. Acad. Sci. USA 89, 11021-11025].
Thus, 8-oxodGTPase activity can be assayed by measuring the
hydrolysis of .alpha.-.sup.32P-labeled 8-oxodGTP to 8-oxodGMP. The
reaction mixture (12.5 .mu.l) contains 20 mM Tris-HCl (pH 8.0), 4
mM MgCl.sub.2, 40 mM NaCl, 20 .mu.M .alpha.-.sup.32P-labeled
8-oxodGTP, 80 .mu.g/ml bovine serum albumin, 8 mM dithiothreitol,
10% glycerol, and a protein extract. The reaction is executed at
30.degree. C. for 20 minutes. Thereafter, an aliquot (2 .mu.l) from
the reaction mixture is spotted onto a PEI-cellulose TLC plate, and
the mixture is fractionated with a solution containing 1 M LiCl for
1 hour. The spots on the TLC plate are then visualized and
quantified by phosphorimaging. The preparation of 8-oxodGTP is
described in Mo et al., ibid.
[0107] An assay for monitoring the activity of AP endonuclease is,
for example, as described by Wilson III, et al. [Wilson III, D. M.,
Takeshita, M., Grollman, A. P., Demple B. (1995) Incision activity
of human apurinic endonuclease (Ape) at abasic site analogs in DNA.
J. Biol. Chem. 270, 16002-16007]. The reaction mixture (10 .mu.l)
contains 50 mM Hepes-KOH pH 7.5, 50 mM KCl, 100 .mu.g/ml bovine
serum albumin, 10 mM MgCl.sub.2, 0.05% Triton X-100, 2 pmol of a
the DNA substrate and a protein extract. Reactions are performed at
37.degree. C. for 5-30 minutes, after which the reaction products
are fractionated by urea-PAGE, to separate the intact and incised
DNA strands. The activity is deduced from the extent of cleavage of
the substrate. The preparation of the substrate is described in the
same reference (Wilson III et al., ibid.).
[0108] An assay for monitoring the activity of deoxyribose
phosphate lyase (dRPase) is, for example, as described by Prasad et
al. [Prasad, R., Beard, W. A., Strauss, P. R. and Wilson, S. H.
(1998) Human DNA polymerase .beta. deoxyribose phosphate lyase.
Substrate specificity and catalytic mechanism. J. Biol. Chem. 273,
15263-15270]. Deoxyribose phosphate lyase (dRPase) activity can be
assayed by following the removal of deoxyribose phosphate from a
.sup.32P 3' end-labeled duplex oligonucleotide containing a
site-specific 5'-incised abasic site. The reaction mixture (10
.mu.l) contains 50 mM Hepes pH 7.4, 2 mM dithiothreitol, 5 mM
MgCl.sub.2, 20 nM .sup.32P-labeled duplex oligonucleotide with a
site specific abasic site (pre-incised at the 5' with AP
endonuclease), and a protein extract. The reaction is carried out
at 37.degree. C. for 15 minutes. After the reaction is terminated,
the product is stabilized by the addition of NaBH.sub.4 to a final
concentration of 340 mM, and incubated for 30 minutes at 0.degree.
C. The DNA is then ethanol precipitated and fractionated by
urea-PAGE. The activity of the dRPase is deduced from the extent of
formation of the shorter reaction product. The preparation of the
DNA substrate is described in the same reference (Prasad et al.,
ibid.).
[0109] Table 3 below lists examples of DNA repair enzymes, the
genes encoding same and the DNA lesion(s) they recognize:
TABLE-US-00003 TABLE 3 Enzyme Gene Substrate 1. Uracil DNA
glycosylase UNG1,2 uracil, 5-fluorouracil, 5-hydroxyuracil
isodialuric acid, alloxan 2. hSMUG1 hSMUG1 uracil 3. hMBD4 hMBD4 U
or T in U/TpG:5meCpG 4. Mismatch-specific thymine/uracil TDG uracil
(U:G), 3,N.sup.4-ethenocytosine DNA glycosylase (eC:G), T (T:G) 5.
Methylpurine DNA glycosylase MPG (ANPG, Aag) 3-methyladenine,
7-methyladenine, 3-methylguanine, 7-methylguanine 8-oxoguanine,
hypoxanthine, 1, N6-ethenoadenine, 1,N2-ethenoguanine 6. hNTH1
(human enodonuclease III hNTH1 thymine glycol, cytosine glycol,
ehomolog) dihydrouracil, formamidopyrimidine urea 7.
Adenine-specific mismatch DNA hMYH A from A:G; A:8-oxoG; C:A
glycosylase (human mutY homolog) 2-hydroxyadenine 8. 8-oxoguanine
DNA glycosylase hOGG1 2,5-amino-5-formamidopyrimidine
7,8-dihydro-8-oxoguanine 9. 8-oxo-GTPase/8-oxodGTPase hMTH1 (NUDT1)
8-oxo-GTP, 8-oxo-dGTP (Human MutT homolog) 10. dUTPase hDUT dUTP
11. AP endonuclease APE1/APE2 abasic site 12. Deoxyribose phosphate
lyase POLB Incised abasic site Enzymes 1-8 are DNA glycosylases;
Enzymes 9 and 10 hydrolyze damaged or unnatural dNTPs, thereby
preventing their incorporation into DNA during DNA synthesis.
Further details concerning mammalian DNA repair genes and activity
can be found in Krokan et al. (2000) FEBS Letters 476: 73-77; and
Wood et al. (2001) Science 291: 1284-1289, both are incorporated
herein by reference.
[0110] The risk according to the present invention can be expressed
in one of a plurality of ways. In one example the risk is expressed
as a fold risk increase in developing cancer as is compared to a
normal, apparently healthy, population, or a reference control
group. In another example, the risk is expressed in enzyme specific
activity units. In another example, a linear or logarithmic risk
scale is generated for either the "fold risk increase" or the
"activity units" and the risk is expressed as a magnitude of the
scale.
[0111] According to still further features in the described
preferred embodiments determining the level of activity of the DNA
repair/damage preventing enzyme is effected using a DNA substrate
having at least one lesion therein.
[0112] As is schematically exemplified by FIGS. 12a-b, a
monomolecular (MMS, FIG. 12a) or plurimolecular (PMS, FIG. 12b)
universal substrate can also be generated and used while
implementing the methods and kits of the present invention. Such a
universal substrate is used according to the present invention to
simultaneously determine the activity of more than a single DNA
repair/damage preventing enzyme. Thus, a universal substrate of the
invention includes at least two (four are shown in FIGS. 12a-b
identified by L1-L4) different DNA lesions specifically recognized
by at least two different DNA repair enzymes. Careful selection of
the positions of the different DNA lesions along the universal
substrate, can be used to ensure the generation of distinguishable
(e.g., size distinguishable) reaction products (P1-P10 in FIG. 12a,
P11-14 in FIG. 12b), being indicative of the activity of the
different DNA repair enzymes. In order to ensure accuracy, the
lesions are selected to be unique to the activities tested. The
length of the universal substrate, especially for a monomolecular
substrate, which preferably includes labels along its length, is
selected such that reciprocal reaction products are substantially
longer than all of the reaction products to be analyzed (P1-P10 in
FIG. 12a). End labeling can be used in the case of a plurimolecular
substrate to circumvent this problem altogether. Thus, the length
of a substrate according to the present invention, without
limitation, can range between 10 base pairs and several hundreds
base pairs.
[0113] A substrate of the invention can thus have at least one
lesion of at least one type or at least one lesion of at least two
types (universal substrate), the lesions preferably being
positioned at predetermined site(s) in the DNA substrate. The
lesion(s) can be of any type, including, but not limited to,
uracil, 5-fluorouracil, 5-hydroxyuracil, isodialuric acid, alloxan,
uracil or thymine in U/TpG:5meCpG, uracil (U:G),
3,N.sup.4-ethenocytosine, (eC:G), T (T:G), 3-methyladenine,
7-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine, 1,
N6-ethenoadenine, 1,N2-ethenoguanine, thymine glycol, cytosine
glycol, dihydrouracil, formamidopyrimidine urea, adenine from A:G;
A:8-oxoG; C:A, 2-hydroxyadenine, 2,5-amino-5-formamidopyrimidine,
7,8-dihydro-8-oxoguanine and abasic site.
[0114] A lesion can be introduced at a unique and defined location
(site) in a DNA molecule using solid phase DNA synthesis, using in
sequence the four conventional phosphoramidite building blocks used
in the synthesis of oligodeoxynucleotides and additional at least
one modified phosphoramidite building block, which when introduced
into the DNA introduces a lesion therein, which lesion is
recognizable by a DNA repair enzyme. In the alternative, a DNA
molecule is exposed to a mutagenic agent (e.g., an oxidative agent
or UV radiation) which forms one or more lesion of one or more
types therein. Even when using this method, one can select a
presubstrate which will result in a product (substrate of the
invention) in which the lesions are non-randomly distributed, since
the extent by which a specific lesion is formed in DNA is often
dependent on the DNA sequence.
[0115] Other alternatives also exist. For example, one can oxidize
a plasmid DNA with an oxidizing agent. This will form several
lesions in the plasmid DNA. One can now use this plasmid DNA to
assay a repair enzyme that acts on this DNA, without knowing
precisely where the lesions are. The enzyme will produce a nick in
the DNA, and this will convert the plasmid from the supercoiled
closed form to the nicked (open circular) form. These two can be
easily distinguished by gel electrophoresis or gradient
centrifugation. In another example a piece of DNA is enzymatically
synthesized in the presence of lesioned building blocks. Other
alternatives are also known, such as chemical deamination, etc.
[0116] Thus, the substrate of the present invention can include at
least two different lesions of at least two types, a single lesion,
or at least two different lesions of a single type.
[0117] A cancer risk determination test according to the present
invention is specifically advantageous for a subject which is known
to be, or is about to be, exposed to environmental conditions
associated with increased risk of developing cancer, such as
smoking and occupational exposure to smoke, ionizing radiation and
other carcinogens.
[0118] As is discussed hereinabove, the effectiveness of cancer
therapy is due to its genotoxic effect affecting cancer cells more
than normal cells. Thus, according to another aspect of the present
invention there is provided a method of predicting the efficacy of
a mutagenic anti-cancer treatment, such as chemotherapy and/or
radiotherapy, in a subject. The method according to this aspect of
the invention is effected by determining a level of activity of a
DNA repair enzyme in a tissue of the subject, and, according to the
level, predicting the efficacy of the mutagenic anti-cancer
treatment in the subject.
[0119] Anti cancer therapy dosage can also be individually
optimized in view of the teachings of the present invention. Thus,
according to still another aspect of the present invention there is
provided a method of selecting dosage of a mutagenic anti-cancer
treatment, such as chemotherapy and/or radiotherapy, for treating a
subject. The method according to this aspect of the invention is
effected by determining a level of activity of a DNA repair/damage
preventing enzyme in a tissue of the subject, and, according to the
level, selecting dosage of the mutagenic anti-cancer treatment for
treating the subject. In this case, the tissue is preferably a
biopsy derived from the cancer itself.
[0120] According to an additional aspect of the present invention
there is provided a kit for determining a level of activity of a
DNA repair/damage preventing enzyme in a tissue of a subject. In
its minimal configuration, the kit includes, a package including,
contained in sealable containers, a DNA substrate having at least
one lesion therein and a reaction buffer selected suitable for
supporting DNA repair activity. Preferably, the kit also includes
test tubes for separating lymphocytes. Preferably, the test tubes
are prepackaged with an anti-coagulant, such as, but not limited
to, heparin. Still preferably, the kit further includes a liquid
having a specific gravity selected effective in separating
lymphocytes from red blood cells via centrifugation, e.g., Ficoll
contained in lymphocytes isolation tubes. Advantageously, the kit
includes a solution having osmolarity selected effective in lysing
red blood cells. In a preferred embodiment of the invention a
protein extraction buffer is also included in the kit. Preferably,
the kit further includes reagents for conducting protein
determinations, e.g., reagents included in the BCA kit by Pierce.
Still preferably, the kit includes a purified DNA repair enzyme,
which serves as a control for such activity.
[0121] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
Examples
[0122] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
Materials and Experimental Methods
[0123] DNA substrates: The DNA substrate was prepared by annealing
two complementary synthetic oligonucleotides, 32-bases long each.
They were synthesized by the Synthesis Unit of the Biological
Services Department at the Weizmann Institute of Science. The
oligonucleotide containing 8-oxoG had the sequence
5'-CCGGTGCATGACACTGTOACC TATCCTCAGCG-3' (SEQ ID NO:1) (0=8-oxoG).
The 8-oxoG phosphoramidite building block was purchased from Glen
Research. The oligonucleotide was .sup.32P-labeled using T4
polynucleotide kinase, and annealed to the oligonucleotide
5'-CGCTGAGGATAGGTCACAGTGTCATGCA CCGG-3' (SEQ ID NO:2). The
radiolabeled duplex was purified by PAGE on a native 10% gel. Its
concentration was determined by the PicoGreen dsDNA quantitation
assay (Molecular Probes).
[0124] Blood samples: Large blood samples were obtained from the
blood bank in the Sheba Medical Center. Samples of 10 ml peripheral
blood were obtained from healthy donors or from cancer patients.
Those were collected after obtaining permission from the
Institutional Helsinki Committee.
[0125] Isolation of peripheral lymphocytes: The blood samples were
processed 18-24 hours after collection. A 100 .mu.l aliquot from
each sample of whole blood was analyzed using a Cobas Micros (Roche
Diagnostic System) blood counter. Ten ml PBS (Dulbecco's phosphate
buffered saline, Sigma) were added to the remaining blood portion,
and peripheral blood lymphocytes were isolated by density gradient
centrifugation of the diluted whole blood on a polysucrose-sodium
metrizoate medium in UNI-SEP tube (NOVAmed, Jerusalem, Israel).
Centrifugation was performed at 1,000.times.g for 30 minutes at
20.degree. C.
[0126] Following centrifugation the lymphocyte band was removed and
washed with PBS buffer. Elimination of red blood cells was done by
lysis in 5 ml of 155 mM NH.sub.4Cl; 0.01 M KHCO.sub.3; 0.1 mM EDTA
for 4 minutes at room-temperature. The lymphocytes were washed with
PBS, and suspended in 1 ml PBS. The number of white blood cells in
this suspension was determined using a Cobas Micros (Roche
Diagnostic System) blood counter.
[0127] Samples containing 1-4.times.10.sup.6 cells were
precipitated by centrifugation at 5,000 rpm, for 4 minutes at room
temperature. The cells pellet was then resuspended to a
concentration of 20,000 cells/.mu.l in 50 mM Tris.HCl (pH 7.1), 1
mM EDTA, 0.5 mM DTT, 0.5 mM spermidine, 0.5 mM spermine, and a
protease inhibitor cocktail (Sigma). The cells were incubated on
ice for 30 minutes, after which they were frozen in liquid
nitrogen. The frozen lymphocytes were stored at -80.degree. C.
[0128] Preparation of a Protein Extract: the Frozen Lymphocytes
were Thawed at 30.degree. C., after which their protein content was
extracted with 220 mM KCl, for 30 minutes on ice. Cell debris was
removed by centrifugation at 13,200 rpm for 15 minutes at 4.degree.
C. Glycerol was added to the protein extract to a final
concentration of 10%, and the extract was frozen in liquid
nitrogen. Protein concentration was determined by the BCA assay kit
(Pierce) using bovine .gamma.-globulin as a standard.
[0129] Standard analysis of OGG activity: The reaction mixture (20
.mu.l) contained 50 mM Tris.HCl (pH 7.1), 1 mM EDTA, 115 mM KCl, 20
.mu.g bovine .gamma.-globulin, 2 pmol PolydA.polydT, 0.5 pmol
substrate and 8-12 .mu.g protein extract. The reaction was carried
out at 37.degree. C. for 30 minutes, after which it was stopped by
the addition of 15 mM EDTA, 0.2% SDS. The proteins were degraded by
incubation with proteinase K (20 .mu.g) for one hour at 37.degree.
C., after which they were treated with 80 mM NaOH for 30 minutes at
37.degree. C. The denatured DNA products were analyzed by
electrophoresis on a 15% polyacrylamide gel containing 8 M urea, in
89 mM Tris.borate, 2.5 mM EDTA pH 8.0, at 1,500 V for 2 hours at
45-50.degree. C. The distribution of radiolabeled DNA products was
visualized and quantified using a Fuji BAS 2500 phosphorimager. One
unit of OGG activity is defined herein to cleave 1 fmol of DNA
substrate in 1 hour at 37.degree. C., under the standard reaction
conditions described herein. In the following, OGGA is presented as
specific activity, i.e., activity units/1 .mu.g of total protein
extract.
[0130] Statistical analysis: A 3-way ANOVA was employed for healthy
subject to compare mean OGGA values, with gender, age (.gtoreq.50,
.ltoreq.50), and smoking status as fixed effects.
[0131] Student's t-test was used to compare the mean OGGA values,
analyzed as a continuous variable, between adenocarcinoma and
squamous cell carcinoma patients.
[0132] To neutralize possible effects on OGGA means originating
from the difference in mean age between the cases and controls,
OGGA means were compared using ANCOVA, with age (treated as a
continuous variable) as a covariate. This analysis was possible
since no significant interaction was found between age and health
conditions.
[0133] Associations were calculated using Fisher's exact test, and
Odds ratios (OR) were calculated from a 2.times.2 table. Adjusted
ORs and CI values were calculated by fitting logistic regression
models with adjustment for age, sex and smoking status for lung
cancer; and adjustment for age only, for lymphoma. OGGA values were
analyzed as a continuous variable or as a dichotomized variable at
values corresponding to 4% (OGGA cutoff at 5.5), 5% (OGGA cutoff at
5.6), 10% (OGGA cutoff at 5.9), 15% (OGGA cutoff at 6.2), 25% (OGGA
cutoff at 6.4) or 50% (OGGA cutoff at 7.3) of the control group.
Age was analyzed as a continuous variable, whereas gender and
smoking status were analyzed as dichotomic variables.
[0134] Odds Ratio (OR) were calculated by the formula (Kleinbaum,
1994)
OR X 1 - X 0 = i = 1 k b i ( X 1 i - X 0 i ) ##EQU00001##
using the b.sub.i estimates from the logistic regression model
where OGGA values were analyzed as a continuous variable
(b.sub.OGGA=-0.624; b.sub.age=0.1; b.sub.smoker=2.9). For example,
X.sub.0, the reference, was used to represent non-smoking, 30
years-old individuals with an OGGA value of 7.0, and X.sub.i was
used to represent the tested subject group. Thus the formula for
the current model is:
ORi=e.sup.-0.624(OGGA.sup.i.sup.-7.0)+0.1(Age.sup.i.sup.-30)+2.9S.sup.i
where OGGA.sub.i and Age.sub.i are the OGGA value and age of
individual i, and S.sub.i is either 1 or zero, for a smoker or a
non smoker, respectively. All the statistical analyses were
performed using SAS software (version 6.12; SAS Institute Inc.,
Cary, N.C.).
Experimental Results
[0135] The OGG activity (OGGA) DNA repair test: Base excision
repair (BER) is initiated by a DNA N-glycosylase, that releases the
damaged or unusual base from DNA, generating an abasic site. The
latter is then repaired by an AP endonuclease (APE/HAP1) and/or the
lyase activity of the glycosylase, as well as the deoxyribose
phosphate lyase (dRPase) activity of DNA polymerase .beta.. The
resulting gap is filled-in by DNA polymerase .beta., forming a
patch of 1-3 nucleotides, followed by ligation (Dianov, et al.,
1992, Singhal, et al., 1995). A long patch pathway of BER was
identified which requires also PCNA, and the FEN-1 flap
endonuclease (Fortini, et al., 1998, Kim, et al., 1998). It was
reported that 8-oxoG can be repaired in cell extracts also by
nucleotide excision repair (Reardon, et al., 1997), however, the in
vivo significance of this finding is not clear (Runger, et al.,
1995). In addition, it was reported that there is
transcription-coupled repair of 8-oxoG, and that it required the
XPG, TFIIH and CSB (Le Page, et al., 2000), and the BRCA1 and BRCA2
proteins (Le Page, et al., 2000).
[0136] While reducing the present invention to practice, an assay
was developed for OGG activity (the OGGA test), using as substrate
a .sup.32P end-labeled synthetic oligonucleotide, 32-base pairs
long, carrying a site-specific 8-oxoG. The source of the OGG
activity was a protein extract prepared from human peripheral blood
lymphocytes (PBL), obtained by Ficoll fractionation from 10 ml
blood samples. A protein extract was prepared from the lymphocytes
by freeze-thaw, followed by salt extraction. The removal of 8-oxoG
from the oligonucleotide, by the OGG activity in the extract,
generates an abasic site, which was rapidly incised either by the
AP lyase activity of the enzyme, or by AP endonucleases present in
the extract. Alkali treatment, which breaks abasic sites, was
performed after the incubation with the extract in order to ensure
complete cleavage of the abasic site, such that only OGG activity
is measured in the test. Analysis by urea-PAGE followed by
phosphorimaging was used to quantify the extent of nicking,
indicated by the formation of a shorter radiolabeled DNA fragment,
17 nucleotides long (FIG. 1a). The OGG activity level (OGGA value)
is measured as specific activity, i.e., units of OGG activity/1
.mu.g of protein extract. One unit of OGG activity cleaves 1 fmol
of DNA substrate in 1 hour at 37.degree. C., under standard
reaction conditions.
[0137] FIGS. 1b-c and 2a-b show a time course and a titration of
OGG activity, respectively, in lymphocyte extracts. The activity
was dependent on the presence of 8-oxoG in the DNA substrate. No
activity was observed when the DNA contained a G instead of the
8-oxoG. This observed activity is mostly due to the OGG1 enzyme,
which was shown to be responsible for most of OGG activity in
extracts prepared from human cells (Monden, et al., 1999). The
existence of OGG2, a second OGG enzyme was reported. However, its
activity was much lower than OGG1 in whole cell extracts (Hazra, et
al., 1998). In addition to OGG, APNG (alkylpurine DNA
N-glycosylase), also termed Aag (alkyladenine DNA glycosylase), or
MPG (N-methylpurine glycosylase), was reported to act on 8-oxoG
(Bessho, et al., 1993) but this finding was challenged in Hang, et
al. (1997). In vivo this protein has no significant role in
removing 8-oxoG from DNA, at least in mice (Engelward, et al.,
1997, Hang, et al., 1997). In order to establish whether MPG is
involved in the removal of 8-oxoG from DNA by lymphocyte extracts,
a competition experiment was performed with an unlabeled duplex
oligonucleotide containing a site-specific hypoxanthine (a
substrate of MPG but not for OGG1; (see, Engelward, et al., 1997,
Hang, et al., 1997)). As can be seen in FIG. 3, this duplex
oligonucleotide did not inhibit the incision of the
8-oxoG-containing DNA by the extract, suggesting that APNG is not
involved in the incision reaction. A control experiment with an
excess of unlabeled duplex oligonucleotide containing a G instead
of 8-oxoG showed no inhibition, whereas a duplex oligonucleotide
containing 8-oxoG-DNA did cause inhibition, as expected (FIGS.
3a-b). These competition experiments are an indication of the
specificity of the OGGA test to 8-oxoG.
[0138] Reproducibility experiments showed that the assay is
accurate and highly reproducible, with a coefficient of variation
.ltoreq.10%. An example of a reproducibility experiment is shown in
Table 4.
TABLE-US-00004 TABLE 4 Reproducibility of the OGGA test A Blood
sample: 1 2 3 4 5 6 7 8 9 10 11 12 OGGA (units/.mu.g protein): 6.8
6.9 6.4 6.5 6.7 7.4 6.9 6.5 6.6 6.7 5.9 6.9 Average OGGA: 6.7
Standard variation: 0.4 Coefficient of variance: 6% Twelve blood
samples from a healthy donor (donor No. 54), 10 ml each, were
processed and assayed for OGGA. One unit of OGG activity incises 1
fmol GO-containing substrate in 60 minutes at 37.degree. C. under
standard assay conditions. B Blood Sample: 1 2 3 4 5 6 Ave SD CV
OGGA (units/ng protein) Experiment 1: 6.7 7.2 6.7 6.8 7.1 6.7 6.9
0.2 3% Experiment 2: 7.9 6.9 7.8 8.2 8.1 7.8 7.8 0.5 6% Experiment
3: 6.9 7.0 7.1 7.3 7.9 7.2 7.2 0.4 5% Overall average OGGA: 7.1
Standard deviation (SD): 0.5 Coefficient of variance (CV): 7% Six
blood samples from a healthy donor (donor No. 50), 10 ml each, were
processed to prepare protein extracts. The table shows the results
of three independent assays performed with these assays on three
different days.
[0139] OGGA value in healthy individuals (control subjects): The
OGGA test was performed on blood samples from 123 healthy
individuals, and the distribution is shown in FIG. 4. The mean OGGA
value was 7.2.+-.1.0 units/.mu.g protein (this will be also dubbed
OGGA value of 7.2.+-.1.0; Table 5).
TABLE-US-00005 TABLE 5 OGGA values in healthy individuals Factor
No. Mean OGGA .+-. SD* P** All 123 7.2 .+-. 1.0 Age, years <50
34 7.6 .+-. 0.9 .gtoreq.50 89 7.0 .+-. 1.0 0.02 Gender Male 53 7.3
.+-. 1.0 Female 70 7.1 .+-. 1.0 0.36 Smoking status Never 88 7.1
.+-. 1.0 Current 35 7.3 .+-. 1.0 0.46 *SD, standard deviation. **P
values are results of 3-way ANOVA.
[0140] The range of OGGA was 3.6-10.1 units/.mu.g protein,
representing a 2.8-fold range of OGG activity. This is a rather
narrow distribution of activity, significantly narrower than
previously reported (Asami, et al., 1996).
[0141] A 3-way ANOVA with gender, age (<50, .gtoreq.50), and
smoking status revealed that there was no significant difference in
mean OGGA value between men (53 individuals; 7.3.+-.1.0 units/.mu.g
protein) and women (70 individuals; 7.1.+-.1.0 units/.mu.g protein;
P=0.36 FIG. 5; Table 5), or between smokers (N=35; 7.3.+-.1.0
units/.mu.g protein) and non-smokers (N=88; 7.1.+-.1.0 units/.mu.g
protein; P=0.46). This indicates that smoking does not affect the
OGGA value in peripheral blood lymphocytes (FIG. 6; Table 5). This
result differs from the result obtained by Asami et al. (1996), who
reported that 8-oxoG repair activity was increased 1.6-fold in
smokers. In contrast, there was a small (6.6%), but statistically
significant decrease in mean OGGA values between the two age
groups: Individuals under the age of 50 had a mean OGGA value of
(7.6.+-.0.9; N=34), whereas those 50 years or older had a mean OGGA
value of (7.0.+-.1.0; N=89; P=0.02; FIG. 7; Table 5). Taken
together these results indicate little or no variation of the OGGA
value with age, smoking status and gender.
[0142] OGGA is not reduced in patients with breast cancer, and is
altered in chronic lymphocytic leukemia (CLL): The OGGA test was
performed on blood samples from 31 breast cancer patients and 19
CLL patients. As can be seen in Table 6, the mean OGGA value was
7.3.+-.1.4 units/.mu.g protein in breast cancer patients, similar
to that of control female subjects (7.1.+-.1.0, P=0.29; Tables 5
and 6). Also, the distribution of OGGA values was similar (FIGS.
8a-d). These results indicate that OGGA is not a risk factor in
breast cancer. The mean OGGA value of CLL patients was 7.9.+-.1.5,
higher than the control subjects (7.2.+-.1.0, P=0.0007; Table 6).
However, the distribution of OGGA values among CLL patients was
similar to the control group (FIGS. 8c-d).
TABLE-US-00006 TABLE 6 Mean OGGA values in cancer patients
Healthy/Disease No. Mean OGGA .+-. SD* P** Healthy 123 7.2 .+-. 1.0
Lung cancer 102 6.0 .+-. 1.5 0.0001 (NSCLC) Breast cancer 31 7.3
.+-. 1.4 0.29 Lymphoma 18 6.2 .+-. 1.8 0.0001 CLL 19 7.9 .+-. 1.5
0.0007 Colorectal cancer 16 7.5 .+-. 1.8 0.047 *SD, standard
deviation. **OGGA means were compared using ANCOVA, with age
(treated as a continuous variable) as a covariate. For breast
cancer patients, the control group consisted of female
subjects.
[0143] OGGA is a risk factor in lung cancer: The OGGA test was
performed with blood samples from 102 patients who suffered from
operable non-small cell lung cancer (NSCLC), and had not been
subjected to either chemo- or radiotherapy at the time when the
blood samples were taken. As can be seen in FIGS. 9a-b and Table 6,
the mean OGGA value was 6.0.+-.1.5 units/.mu.g protein,
significantly lower than the mean value of controls (7.2.+-.1.0,
P=0.0001). Analyzing separately cases with adenocarcinoma or
squamous cell carcinoma revealed a similar OGGA level in these two
main sub-types of NSCLC: adenocarcinoma: 6.1.+-.1.5, N=37; squamous
cell carcinoma: 5.8.+-.1.6 N=35; P=0.44 (the other 30 cases were
either other sub-types or unclassified NSCLC). The comparison of
the distributions of OGGA in controls and in cases highlights the
difference between the two groups. As can be clearly seen in FIGS.
9a-b, there is a shift to lower OGGA values in cases as compared to
controls. For example, only 4% of controls have OGGA values of
.ltoreq.5.5, whereas 38% of cases have OGGA values in this range.
This includes values 2-3 fold lower than the mean OGGA values of
the controls. The mean age of the control group (57.+-.14) was
significantly different from the cases group (68.+-.10;
P<0.0001). Therefore, logistic regression, adjusted for age, was
used to analyze associations, and analysis of covariance was used
to compare age-adjusted mean OGGA values.
[0144] To analyze the association between levels of OGGA and
presence of lung cancer logistic regression was used, where the
binary dependent variable was presence/absence of lung cancer, and
with age as a continuous variable, and gender, smoking status and
OGGA as dichotomic variables. The latter was dichotomized at values
corresponding to 5% (OGGA cutoff at 5.6), 10% (OGGA cutoff at 5.9),
15% (OGGA cutoff at 6.2), 25% (OGGA cutoff at 6.4) or 50% (OGGA
cutoff at 7.3) of the control group. As can be seen in Table 7,
smoking is strongly associated with lung cancer, in agreement with
its established role as a major risk factor in the disease. For
example, in a model where OGGA was dichotomized at .ltoreq.5.9
(corresponding to 10% of the controls), the Odds Ratio (OR) for
smokers was 20.8 (95% CI 7.8-55.4, P=0.0001). In a model where the
OGGA cutoff was defined as .ltoreq.7.3 (corresponding to 50% of
controls), the OR for smokers was 23.0 (95% CI 8.9-59.2, P=0.0001).
The gender had no significant effect in any of the models, whereas
increased age was associated with the presence of lung cancer.
Notice that although the OR for age was relatively small in all
models (1.1 95% CI 1.1-1.2, P=0.0001) it is statistically
significant. The age was analyzed as a continuous variable, and the
relatively small OR is given per one-year change. Therefore, its
final effect when applied to a particular change of age might be
much larger (see Table 8 below).
[0145] As can be seen from Table 7, a clear association was found
between the level of OGGA and presence of lung cancer. Moreover,
there is a dose-dependent effect, with higher OR obtained for lower
OGGA. For example, OR values of 3.9, 5.2, 7.0 and 9.0 were obtained
for cutoff OGGA values of 7.3, 6.4, 5.9 and 5.6, respectively
(Table 7). These high OR values indicate a strong association
between low OGGA and lung cancer. Moreover, the increase in OR with
decreasing OGGA further strengthens the significance of the
association. In addition, the high OR values argue against the
possibility of a selection bias in the control group.
TABLE-US-00007 TABLE 7 Association of low OGGA and lung cancer
Adjusted Odds Ratio (95% CI) * OGGA DNA repair cutoff .sup..dagger.
Controls Cases OGGA Smoking Age Sex .ltoreq.5.6 (5%) 7 42 9.0
(3.2-25.0) 18.6 (7.1-48.7) 1.1 (1.1-1.2) 1.0 (0.4-2.1) >5.6 116
60 P = 0.0001 P = 0.0001 P = 0.0001 P = 0.79 .ltoreq.5.9 (10%) 14
52 7.0 (3.0-16.7) 20.8 (7.8-55.4) 1.1 (1.1-1.2) 1.0 (0.4-2.2)
>5.9 109 50 P = 0.0001 P = 0.0001 P = 0.0001 P = 0.96
.ltoreq.6.2 (15%) 18 60 6.5 (2.9-14.5) 21.5 (8.0-58.0) 1.1
(1.1-1.2) 0.9 (0.4-2.0) >6.2 105 42 P = 0.0001 P = 0.0001 P =
0.0001 P = 0.77 .ltoreq.6.4 (20%) 25 63 5.2 (2.4-11.2) 20.6
(7.9-53.6) 1.1 (1.1-1.2) 0.9 (0.4-2.0) >6.4 98 39 P = 0.0001 P =
0.0001 P = 0.0001 P = 0.77 .ltoreq.6.6 (25%) 34 68 4.3 (2.0-9.0)
21.4 (8.2-55.5) 1.1 (1.1-1.2) 0.9 (0.4-2.0) >6.6 89 34 P =
0.0002 P = 0.0001 P = 0.0001 P = 0.85 .ltoreq.7.3 (50%) 63 83 3.9
(1.7-8.6) 23.0 (8.9-59.2) 1.1 (1.1-1.2) 0.9 (0.4-1.8) >7.3 60 19
P = 0.0009 P = 0.0001 P = 0.0001 P = 0.67 The logistic regression
model is based on age as a continuous variable, and the dichotomic
variables were smoking status (smoker, non-smoker), gender (female,
male) and DNA repair activity value (low or normal, with various
cutoff values, as indicated). The goodness of fit of the model, as
described by R.sup.2, is in the range of 58-61%. * 95% CI, 95%
confidence interval. .sup..dagger. Cutoff values defined for the
dichotomic variable of OGGA. The numbers in parentheses show the
corresponding percentage of control subjects with OGGA values lower
than or equal to the cutoff value.
[0146] In case-control studies there is a possibility that the
examined variable is a consequence of the disease, rather than
being a risk factor. In the present case, the possibility that the
lung tumor causes a decrease of OGGA in peripheral blood
lymphocytes (PBL) was considered. The OGGA value may be affected,
for example, by factors that the tumor secretes into the blood
stream. The main treatment of NSCLC is surgical removal of the
tumor. This offers a way to distinguish between a causative and a
resultive model for the association of PBL OGGA and lung cancer.
Once eliminated from the lung, the effect the tumor have (if any)
on OGGA in lymphocytes should decay with time. No correlation
between OGGA and the time period that passed between surgery and
taking the blood sample (ranging from 4 months before surgery to
over a year after surgery) was found, indicating that whether the
samples were taken before or after surgery had no effect on the
level of OGGA in PBL. In the current group of case subjects, most
(67/102) samples were taken after surgery. These results, clearly
indicate that reduced OGGA is indeed a risk factor in lung
cancer.
[0147] The simplest biological explanation for the present finding
is the following: Low OGGA in PBL reflects low OGGA in the lungs.
Correlations between DNA repair activities in PBL and lung cells
(Auckley et al., 2001) or gastric mucosa (Kyrtopoulos et al., 1990)
were previously reported. The lower DNA repair capacity leads to a
reduced ability to repair oxidative DNA damage, and as a result
8-oxoguanine accumulates and leads to an increased mutation rate,
which causes a higher cancer risk. In smokers there is an overload
of DNA damage in the lungs, and therefore a higher risk is
expected. No interaction was found between OGGA values and smoking
status, implying that each of the two is an independent risk factor
for lung cancer. This means that low OGGA is a risk factor also in
non-smokers. This is not surprising, since oxidative DNA damage is
a common intracellular damage that occurs even without exposure to
external agents (Lindahl, 1993).
[0148] As discussed before, OGGA is not reduced in patients with
breast cancer. This suggests that the repair of 8-oxoG is a
bottleneck in the case of lung cancer, and in some additional
cancers, but not in others (e.g., breast cancer). This is
consistent with the finding that hereditary defects in particular
DNA repair genes cause predisposition to specific types of cancer.
For example, defects in nucleotide excision repair were shown to
cause skin cancer (Weeda et al., 1993) whereas defects in mismatch
repair cause hereditary non-polyposis colon cancer (Modrich, 1994).
To our knowledge the results presented herein, are the first
demonstration that decreased activity of a specific base excision
repair enzyme is associated with cancer.
[0149] A useful application of the results of this study would be a
quantitative model, which will provide an estimation of the risk of
lung cancer associated with a particular OGGA value, age and
smoking status. For diseases that do not occur frequently, such as
lung cancer, and assuming that the cases and the controls are
reasonably representative of the population, the odds ratio can be
used as estimated relative risk (Gordis, 1996). Thus, a model was
formulated using logistic regression, with age and OGGA as
continuous variables, and smoking status as a dichotomic variable
(smokers or non-smokers). This yielded OR values for lung cancer
and these were taken as an estimation of risk. The OR values were
calculated by dividing the odds of each particular group (having a
particular age, OGGA value and smoking status) by the odds of 30
years-old non-smokers with a normal OGGA value of 7.0 (the
reference group; OR of 1.0). The OR values for a specific age, OGGA
value and smoking status are listed in Table 8. For example,
according to Table 8, the estimated risk for 30 years-old smokers
with a low OGGA value of 4.0, is 118-fold higher than the
reference. At the age of 40, the estimated risk will increase to
321-fold higher than the reference. This high estimated risk is
primarily the combined result of smoking and low OGGA. Having a low
repair activity to start with, smoking causes further overloading
of DNA damage, therefore leading to a high cancer risk. This model
is instrumental in clarifying the fact that the combination of
smoking and low OGGA causes a dramatic increase in susceptibility
to lung cancer. For example, 40 years-old non-smokers with an OGGA
value of 4.0 have an estimated risk 18-fold higher than the
reference, compared to an estimated risk of 321-fold higher than
the reference of smokers with the same age and OGGA (Table 8).
[0150] The OGGA test can be used to screen smokers for reduced DNA
repair capacity. These individuals can be persuaded to quit smoking
based on their personal reduced ability to cope with DNA damage.
Since smoking is the main contributor to the high relative
estimated risk for lung cancer (Tables 7 and 8), quitting smoking
is expected to significantly improve the chances of preventing lung
cancer. Such an approach of personalized smoking cessation, based
on personal susceptibility, may provide a successful and
cost-effective strategy to prevent lung cancer, and may be extended
to include additional DNA repair assays.
TABLE-US-00008 TABLE 8 An odds ratio model for estimating the risk
of lung cancer for specific DNA repair OGGA values, age and smoking
status Estimated Risk OGGA (Odds Ratio*) Age, y value Non-smokers
Smokers 30 7 1 18 30 6 2 34 30 5 3 63 30 4 7 118 30 3 12 221 40 7 3
49 40 6 5 92 40 5 9 172 40 4 18 321 40 3 33 599 50 7 7 134 50 6 14
251 50 5 26 468 50 4 48 873 50 3 90 1629 60 7 20 365 60 6 37 681 60
5 70 1272 60 4 131 2373 60 3 244 4429 70 7 55 992 70 6 102 1852 70
5 190 3456 70 4 355 6451 70 3 662 12040 *The Table is based on
logistic regression analysis of the case-control study, and
therefore the numbers represent only estimated values of risk. The
odds ratio is calculated relative to the odds ratio of 30 years-old
non-smokers with an OGGA value of 7.0.
[0151] The data presented herein indicates that low OGGA is a risk
factor for lung cancer also among non-smokers (Tables 7 and 8).
What can non-smokers with low OGGA do to protect themselves? One
possibility is to make sure that they are not exposed to external
sources of oxidative DNA damage such as secondary smoking or
ionizing radiation. The latter includes radiology departments in
hospitals, nuclear industry, and nuclear reactors. However,
oxidative DNA damage is caused also by internal agents; therefore,
dietary anti-oxidants might have a protective effect. Large
population studies found that antioxidants had no protective effect
against cancer (reviewed in Collins, 1999; Lippman and Spitz,
2001). However, these food additives might have a protective effect
when taken by individuals with low capacity to repair oxidative DNA
damage.
[0152] Low OGG activity is a risk factor in lymphoma: Analysis of
18 lymphoma patients showed a clear shift to lower OGG DNA repair
values (FIGS. 10a-b; Table 6): The mean OGGA value was 6.2.+-.1.8
units/.mu.g protein, significantly lower than in healthy
individuals (P=0.0001). Analysis of Normal and Low repair in
healthy individuals and in lymphoma patients using logistic
regression yielded an adjusted Odds Ratio of 15.2 (95% CI,
3.7-62.5). This means that after adjustment for age, lymphoma
patients were 15 times more likely than the healthy controls to
have a Low OGGA. This indicates that Low OGGA is a risk factor in
lymphoma (Table 9).
TABLE-US-00009 TABLE 9 Association of Low OGGA and lymphoma Factor
Crude OR Adjusted** OR OGGA Cases Controls (95% CI*) (95% CI)
Normal >5.5 12 118 Low .ltoreq.5.5 6 5 11.8 (3.1-44.5) 15.2
(3.7-62.5) P = 0.0006 P = 0.0002 *CI, 95% confidence interval.
**Adjusted for age.
[0153] OGG activity seems to be reduced in colorectal cancer
patients: An analysis was performed with 16 colorectal cancer
patients (FIGS. 11a-b). Two of the patients exhibited low OGG
(12%). This data indicates that low OGG is a risk factor in
colorectal cancer.
[0154] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0155] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
CITED REFERENCES
Additional References are Cited in the Text
[0156] Aburatani, H., Y. Hippo, T. Ishida, R. Takashima, C.
Matsuba, T. Kodama, M. Takao, A. Yasui, K. Yamamoto and M. Asano.
(1997) Cloning and characterization of mammalian
8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic
lyase, a functional mutM homologue, Cancer Res., 57, 2151-2156.
[0157] Arai, K., K. Morishita, K. Shinmura, T. Kohno, S. R. Kim, T.
Nohmi, M. Taniwaki, S. Ohwada and J. Yokota. (1997) Cloning of a
human homolog of the yeast OGG1 gene that is involved in the repair
of oxidative damage, Oncogene, 14, 2857-2861. [0158] Asami, S., T.
Hirano, R. Yamaguchi, Y. Tomioka, H. Itoh and H. Kasai (1996)
Increase of a type of oxidative DNA damage, 8-hydroxyguanine, and
its repair activity in human leukocytes by cigarette smoking,
Cancer Res., 56, 2546-2549. [0159] Asami, S., H. Manabe, J. Miyake,
Y. Tsurudome, T. Hirano, R. Yamaguchi, H. Itoh and H. Kasai. (1997)
Cigarette smoking induces an increase in oxidative DNA damage,
8-hydroxydeoxyguanosine, in a central site of the human lung,
Carcinogenesis, 18, 1763-1766. [0160] Athas, W. F., M. A. Hedayati,
G. M. Matanoski, E. R. Farmer and L. Grossman. (1991) Development
and field-test validation of an assay for DNA repair in circulating
human lymphocytes, Cancer Res., 51, 5786-5793. [0161] Aucklley, D.
H., Crowell, R. E., Heaphy, E. R., Stidley, C. A., Lechner, J. F.,
Gilliland, F. D. and A., B. S. (2001) Reduced DNA-dependent prorein
kinase is associated with lung cancer. Carcinogenesis, 22, 723-727.
[0162] Audebert, M., S. Chevillard, C. Levalois, G. Gyapay, A.
Vieillefond, J. Klijanienko, P. Vielh, A. K. El Naggar, S. Oudard,
S. Boiteux and J. P. Radicella. (2000) Alterations of the DNA
repair gene OGG1 in human clear cell carcinomas of the kidney,
Cancer Res., 60, 4740-4744. [0163] Bessho, T., R. Roy, K. Yamamoto,
H. Kasai, S, Nishimura, K. Tano and S. Mitra. (1993) Repair of
8-hydroxyguanine in DNA by mammalian N-methylpurine-DNA
glycosylase, Proc. Natl. Acad. Sci. USA, 90, 8901-8904. [0164]
Bishop, J. M. (1995) Cancer: The rise of the genetic paradigm,
Genes & Dev., 9, 1309-1315. [0165] Bjoras, M., L. Luna, B.
Johnsen, E. Hoff, T. Haug, T. Rognes and E. Seeberg. (1997)
Opposite base-dependent reactions of a human base excision repair
enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites,
EMBO J., 16, 6314-6322. [0166] Chevillard, S., J. P. Radicella, C.
Levalois, J. Lebeau, M. F. Poupon, S. Oudard, B. Dutrillaux and S.
Boiteux. (1998) Mutations in OGG1, a gene involved in the repair of
oxidative DNA damage, are found in human lung and kidney tumours,
Oncogene, 16, 3083-3086. [0167] Collins, A. R. (1999) Oxidative DNA
damage, antioxidants, and cancer. BioEssays, 21, 238-246. Connor,
F., D. Bertwistle, P. J. Mee, G. M. Ross, S. Swift, E. Grigorieva,
V. L. J. Tybulewicz and A. Ashworth. (1997) Tumorigenesis and a DNA
repair defect in mice with a truncating Brca2 mutation, Nature
Genet, 17, 423-430. [0168] Dianov, G., A. Price and T. Lindahl.
(1992) Generation of single-nucleotide repair patches following
excision of uracil residues from DNA, Mol. Cell Biol., 12,
1605-1612. [0169] Echols, H. and M. F. Goodman. (1991) Fidelity
mechanisms in DNA replication, Annu. Rev. Biochem., 60, 477-511.
[0170] Engelward, B. P., G. Weeda, M. D. Wyatt, J. L. Broekhof, J.
de Wit, I. Donker, J. M. Allan, B. Gold, J. H. Hoeijmakers and L.
D. Samson. (1997) Base excision repair deficient mice lacking the
Aag alkyladenine DNA glycosylase, Proc. Natl. Acad. Sci. USA, 94,
13087-13092. [0171] Fishel, R., M. K. Lescoe, M. R. Rao, N. G.
Copeland, N. A. Jenkins, J. Garber, M. Kane and R. Kolodner. (1993)
The human mutator gene homolog MSH2 and its association with
hereditary nonpolyposis colon cancer, Cell, 75, 1027-38. [0172]
Fortini, P., B. Pascucci, E. Parlanti, R. W. Sobol, S. H. Wilson
and E. Dogliotti. (1998) Different DNA polymerases are involved in
the short- and long-patch base excision repair in mammalian cells,
Biochemistry, 37, 3575-3580. [0173] Friedberg, E. C., G. C. Walker
and W. Siede (1995) DNA repair and mutagenesis, ASM Press,
Washington, D.C. [0174] Gajewski, E., G. Rao, Z. Nackerdien and M.
Dizdaroglu. (1990) Modification of DNA bases in mammalian chromatin
by radiation-generated free radicals, Biochemistry, 29, 7876-7882.
[0175] Gordis, L. (1996) Epidemiology, W.B. Saunders Co.,
Philadelphia. [0176] Gowen, L. C., A. V. Avrutskaya, A. M. Latour,
B. H. Koller and S. A. Leadon. (1998) BRCA1 required for
transcription-coupled repair of oxidative DNA damage, Science, 281,
1009-1012. [0177] Hanawalt, P. C. (1994) Transcription-coupled
repair and human disease, Science, 266, 1957-1958. [0178] Hang, B.,
B. Singer, G. P. Margison and R. H. Elder. (1997) Targeted deletion
of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,
N6-ethenoadenine and hypoxanthine but not 3, N4-ethenocytosine or
8-oxoguanine, Proc. Natl. Acad. Sci. USA, 94, 12869-12874. [0179]
Hazra, T. K., T. Izumi, L. Maidt, R. A. Floyd and S. Mitra. (1998)
The presence of two distinct 8-oxoguanine repair enzymes in human
cells: their potential complementary roles in preventing mutation.,
Nucleic Acids Res., 26, 5116-5122. [0180] Helzlsouer, K. J., E. L.
Harris, R. Parshad, H. R. Perry, F. M. Proce and K. K. Sanford.
(1996) DNA repair proficiency: Potential susceptibility factor for
breast cancer, J. Natl. Cancer. Inst., 88, 754-755. [0181]
Hernandez-Boussard, T., P. Rodriguez-Tome, R. Montesano and P.
Hainaut, P. (1999) IARC p53 mutation database: a relational
database to compile and analyze p53 mutations in human tumors and
cell lines. International Agency for Research on Cancer, Hum.
Mutat., 14, 1-8. [0182] Hollstein, M., Shomer, B., Greenblatt, M.,
Soussi, T., Hovig, E., Montesano, R. and Harris, C. C. (1996)
Somatic point mutations in the p53 gene of human tumors and cell
lines: updated compilation. Nucleic Acids Res., 24, 141-146. [0183]
Hutchinson, F. (1985) Chemical changes induced in DNA by ionizing
radiation, Prog. Nucleic Acid Res. Mol. Biol., 32, 115-154. [0184]
Hyun, J. W., J. Y. Choi, H. H. Zeng, Y. S. Lee, H. S. Kim, S. H.
Yoon and M. H. Chung. (2000) Leukemic cell line, KG-1 has a
functional loss of hOGG1 enzyme due to a point mutation and
8-hydroxydeoxyguanosine can kill KG-1, Oncogene, 19, 4476-4479.
[0185] Ishida, T., Y. Hippo, Y. Nakahori, I. Matsushita, T. Kodama,
S, Nishimura and H. Aburatani. (1999) Structure and chromosome
location of human OGG1, Cytogenet. Cell Genet., 85, 232-236. [0186]
Jyothish, B., R. Ankathil, B. Chandini, B. Vinodkumar, G. Sunil
Nayar, D. Dinesh Roy, J. Madhavan and M. Krishnan Nair. (1998) DNA
repair proficiency: a potential marker for identification of high
risk members in breast cancer families, Cancer Lett., 124, 9-13.
[0187] Kim, K., S. Biade and Y. Matsumoto. (1998) Involvement of
flap endonuclease 1 in base excision DNA repair, J. Biol. Chem.,
273, 8842-8848. [0188] Kleinbaum, D. G. (1994) Logistic Regression,
Springer-Verlag, New York. [0189] Klungland, A., I. Rosewell, S.
Hollenbach, E. Larsen, G. Daly, B. Epe, E. Seeberg, T. Lindahl and
D. E. Barnes. (1999) Accumulation of premutagenic DNA lesions in
mice defective in removal of oxidative base damage, Proc. Natl.
Acad. Sci. USA, 96, 13300-13305. [0190] Kyrtopoulos, S. A.,
Ampatzi, P., Davaris, P., Haritopoulos, N. and Golematis, B. (1990)
Studies in gastric carcinogenesis. IV. O6-methyguanine and its
repair in normal and atrophic biopsy specimen of human gastric
mucosa. Correlation of O6-methylguanine-DNA alkyltransferase
activities in gastric mucosa and circulating lymphocytes.
Carcinogenesis, 11, 431-436. [0191] Laval, F. (1994) Expression of
the E. coli fpg gene in mammalian cells reduces mutagenicity of g
rays, Nucleic Acids Res., 22, 4943-4946. [0192] Le Page, F., E. E.
Kwoh, A. Avrutskaya, A. Gentil, S. A. Leadon, A. Sarasin and P. K.
Cooper. (2000) Transcription-coupled repair of 8-oxoguanine:
requirement for XPG, TFIIH, and CSB and implications for Cockayne
syndrome, Cell, 101, 159-171. [0193] Le Page, F., V.
Randrianarison, D. Marot, J. Cabannes, M. Perricaudet, J. Feunteun
and A. Sarasin. (2000) BRCA1 and BRCA2 are necessary for the
transcription-coupled repair of the oxidative 8-oxoguanine lesion
in human cells, Cancer Res., 60, 5548-5552. [0194] Leach, F. S., N.
C. Nicolaides, N. Papadopoulos, B. Liu, J. Jen, R. Parsons, P.
Peltomaki, P. Sistonen, L. A. Aaltonen, M. Nystromlahti, X. Y.
Guan, J. Zhang, P. S. Meltzer, J. W. Yu, F. T. Kao, D. J. Chen, K.
M. Cerosaletti, R. E. K. Fournier, S. Todd, T. Lewis, R. J. Leach,
S. L. Naylor, J. Weissenbach, J. P. Mecklin, H. Jarvinen, G. M.
Petersen, S. R. Hamilton, J. Green, J. Jass, P. Watson, H. T.
Lynch, J. M. Trent, J. M. Trent, A. de la Chapelle, K. W. Kinzler
and B. Vogelstein. (1993) Mutations of a mutS homolog in hereditary
nonpolyposis colorectal cancer, Cell, 75, 1215-1225. [0195]
Leanderson, P. and C. Tagesson. (1992) Cigarette smoke-induced DNA
damage in cultured human lung cells: role of hydroxyl radicals and
endonuclease activation, Che. Biol. Interact., 81, 197-208. [0196]
Lindahl, T. (1993) Instability and decay of the primary structure
of DNA. Nature, 362, 709-715. [0197] Lippman, S. M. and Spitz, M.
R. (2001) Lung cancer chemoprevention: An integrated approach. J.
Clin. Oncol., 19, 74s-82s. [0198] Livneh, Z., O. Cohen-Fix, R.
Skaliter and T. Elizur. (1993) Replication of damaged DNA and the
molecular mechanism of ultraviolet light mutagenesis, CRC Crit.
Rev. Biochem. Mol. Biol., 28, 465-513. [0199] Lu, R., H. M. Nash
and G. L. Verdine. (1997) A mammalian DNA repair enzyme that
excises oxidatively damaged guanines maps to a locus frequently
lost in lung cancer., Curr. Biol., 7, 397-407. [0200] Maki, H. and
M. Sekiguchi. (1992) MutT protein specifically hydrolyses a potent
mutagenic substrate for DNA synthesis, Nature (London), 355,
273-275. [0201] Mattson, M. E., Pollack, E. S. & Cullen, J. W.
(1987) What are the odds that smoking will kill you? Am. J. Public
Health 77, 425-431. [0202] Minna, J. D., Roth, J. A. and Gazdar, A.
F. (2002) Focus on lung cancer. Cancer Cell, 1, 49-52. [0203]
Minowa, O., T. Arai, M. Hirano, Y. Monden, S, Nakai, M. Fukuda, M.
Itoh, H. Takano, Y. Hippou, H. Aburatani, K. Masumura, T. Nohmi, S,
Nishimura and T. Noda. (2000) Mmh/Ogg1 gene inactivation results in
accumulation of 8-hydroxyguanine in mice, Proc. Natl. Acad. Sci.
USA, 97, 4156-4161. [0204] Modrich, P. (1994) Mismatch repair,
genetic stability, and cancer, Science, 266, 1959-1960. [0205]
Monden, Y., T. Arai, M. Asano, E. Ohtsuka, H. Aburatani and S,
Nishimura. (1999) Human MMH (OGG1) Type 1a Protein Is a Major
Enzyme for Repair of 8-Hydroxyguanine Lesions in Human Cells,
Biochem. Biophys. Res. Commun., 258, 605-610. [0206] Parshad, R.,
F. M. Price, V. A. Bohr, K. H. Cowans, J. A. Zujewski and K. K.
Sanford. (1996) Deficient DNA repair capacity, a predisposing
factor in breast cancer, Brit. J. Cancer, 74, 1-5. [0207] Parsons,
R., G. M. Li, M. J. Longley, W. H. Fang, N. Papadopoulos, J. Jen,
I. C. A. de, K. W. Kinzler, B. Vogelstein and P. Modrich. (1993)
Hypermutability and mismatch repair deficiency in RER+tumor cells,
Cell, 75, 1227-36. [0208] Patel, K. J., V. P. C. C. Yu, H. Lee, A.
Corcoran, F. C. Thistlethwaite, M. J. Evans, W. H. Colledge, L. S.
Friedman, B. A. J. Ponder and A. R. Venkitaraman. (1998)
Involvement of Brca2 in DNA Repair, Molecular Cell, 1, 347-357.
[0209] Patel, R. K., A. H. Trivedi, D. C. Arora, J. M. Bhatavdekar
and D. D. Patel. (1997) DNA repair proficiency in breast cancer
patients and their first-degree relatives, Int. J. Cancer, 73,
20-24. [0210] Pavlov, Y. I., D. T. Minnick, S. Izuta and T. A.
Kunkel. (1994) DNA replication fidelity with 8-oxodeoxyguanosine
triphosphate, Biochemistry, 33, 4695-4701. [0211] Radicella, J. P.,
C. Dherin, C. Desmaze, M. S. Fox and S. Boiteux. (1997) Cloning and
characterization of hOGG1, a human homolog of the OGG1 gene of
Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. USA, 94,
8010-8015. [0212] Reardon, J. T., T. Bessho, H. C. Kung, P. H.
Bolton and A. Sancar. (1997) In vitro repair of oxidative DNA
damage by human nucleotide excision repair system: possible
explanation for neurodegeneration in xeroderma pigmentosum
patients, Proc. Natl. Acad. Sci. USA, 94, 9463-9468. [0213]
Roldan-Arjona, T., Y. F. Wei, K. C. Carter, A. Klungland, C.
Anselmino, R. P. Wang, M. Augustus and T. Lindahl. (1997) Molecular
cloning and functional expression of a human cDNA encoding the
antimutator enzyme 8-hydroxyguanine DNA glycosylase, Proc. Natl.
Acad. Sci. USA, 94, 8016-8020. [0214] Rosenquist, T. A., D. O.
Zharkov and A. P. Grollman (1997) Cloning and characterization of a
mammalian 8-oxoguanine DNA glycosylase, Proc. Natl. Acad. Sci. USA,
94, 7429-7434. [0215] Runger, T. M., B. Epe and K. Moller. (1995)
Repair of ultraviolet B and singlet oxygen-induced DNA damage in
xeroderma pigmentosum cells, J. Invest. Dermatol., 104, 68-73.
[0216] Sagher, D., T. Karrison, J. L. Schwartz, R. Larson, P. Meier
and B. Strauss. (1988) Low O6-alkylguanine DNA alkyltransferase
activity in the peripheral blood lymphocytes of patients with
therapy-related acute nonlymphocytic leukemia, Cancer Res., 48,
3084-3089. [0217] Sancar, A. (1994) Mechanisms of DNA repair,
Science, 266, 1954-1956. [0218] Savitsky, K., A. Bar-Shira, S.
Gilad, G. Rotman, Y. Ziv, L. Vanagaite, D. A. Tagle, S. Smith, T.
Uziel, S. Sfez, M. Ashkenazi, I. Pecker, M. Frydman, R. Harnik, S.
R. Patanjali, A. Simmons, G. A. Clines, A. Sartiel, R. A. Gatti, L.
Chessa, O, Sanal, M. Lavin, N. G. J. Jaspers, A. M. R. Taylor, C.
F. Arlett, T. Miki, S. M. Weissman, M. Lovett, F. S. Collins and Y.
Shiloh. (1995) A single Ataxia Telangiectasia gene with a product
similar to PI-3 kinase, Science, 268, 1749-1753. [0219] Scully, R.,
J. Chen, A. Plug, Y. Xiao, D. Weaver, J. Feunteun, T. Ashley and D.
M. Livingston. (1997) Association of BRCA1 with Rad51 in Mitotic
and Meiotic cells, Cell, 88, 265-275. [0220] Sharan, S. K., M.
Morimatsu, U. Albrecht, D. S. Lim, E. Regel, C. Dinh, A. Sands, G.
Eichele, P. Hasty and A. Bradley. (1997) Embryonic lethality and
radiation hypersensitivity mediated by Rad51 in mice lacking Brca2,
Nature, 386, 804-810. [0221] Shibutani, S., M. Takeshita and A. P.
Grollman (1991) Insertion of specific bases during DNA synthesis
past the oxidation-damaged base 8-oxodG, Nature, 349, 431-434.
[0222] Shinmura, K., T. Kohno, H. Kasai, K. Koda, H. Sugimura and
J. Yokota. (1998) Infrequent mutations of the hOGG1 gene, that is
involved in the excision of 8-hydroxyguanine in damaged DNA, in
human gastric cancer, Jpn. J. Cancer Res., 89, 825-828. [0223]
Singhal, R. K., R. Prasad and S. H. Wilson. (1995) DNA polymerase
beta conducts the gap-filling step in uracil-initiated base
excision repair in a bovine testis nuclear extract, J Biol Chem,
270, 949-957. [0224] Srivastava, S., Z. Zou, K. Pirollo, W.
Blattner and E. H. Chang. (1990) Germ-line transmission of a
mutated p53 in a cancer-prone family with Li-Fraumeni syndrome,
Nature (London), 348, 747-749.
[0225] Strauss, B. S. (1985) Translesion DNA synthesis: polymerase
response to altered nucleotide, Cancer Surv., 4, 493-516. [0226]
Vandenbroucke, J. P., T. Koster, E. Briet, P. H. Reitsma, R. M.
Bertina and F. R. Rosendaal. (1994) Increased risk of venous
thrombosis in oral-contraceptive users who are carriers of factor V
Leiden mutation, Lancet, 344, 1453-1457. [0227] Vogelstein, B. and
K. W. Kinzler. (1993) The multistep nature of cancer, Trends
Genet., 9, 138-141. [0228] Weeda, G., J. H. J. Hoeijmakers and D.
Bootsma. (1993) Genes controlling nucleotide excision repair in
eukaryotic cells, BioEssays, 15, 249-258. [0229] Wei, Q., L. Cheng,
W. K. Hong and M. R. Spitz. (1996) Reduced DNA repair capacity in
lung cancer patients, Cancer Res., 56, 4103-4107. [0230] Wei, Q.,
G. M. Matanoski, E. R. Farmer, M. A. Hedayati and L. Grossman.
(1993) DNA repair and aging in basal cell carcinoma: a molecular
epidemiology study, Proc. Natl. Acad. Sci. USA, 90, 1614-1618.
[0231] Wei, Q., G. M. Matanoski, E. R. Farmer, M. A. Hedayati and
L. Grossman. (1994) DNA repair and susceptibility to basal cell
carcinoma: a case-control study, Am. J. Epidemiol., 140, 598-607.
[0232] Weinberg, R. A. (1989) Oncogenes, antioncogenes, and the
molecular bases of multistep carcinogenesis, Cancer Res., 49,
3713-3721. [0233] Wikman, H., A. Risch, F. Klimek, P. Schmezer, B.
Spiegelhalder, H. Dienemann, K. Kayser, V. Schulz, P. Drings and H.
Bartsch. (2000) hOGG1 polymorphism and loss of heterozygosity
(LOH): significance for lung cancer susceptibility in a caucasian
population, Int. J. Cancer, 88, 932-937. [0234] Wood, M. L., M.
Dizdaroglu, E. Gajewski and J. M. Essigmann. (1990) Mechanistic
studies of ionizing radiation and oxidative mutagenesis: Genetic
effects of a single 8-hydroxyguanine (7-hydro-8-oxo-guanine)
residue inserted at a unique site in a viral genome, Biochemistry,
29, 7024-7032.
Sequence CWU 1
1
14132DNAArtificial sequencesynthetic oligonucleotide 1ccggtgcatg
acactgtnac ctatcctcag cg 32232DNAArtificial sequencesynthetic
oligonucleotide 2cgctgaggat aggtcacagt gtcatgcacc gg 3232062DNAHomo
sapiens 3ctcccagccc gtctccccgc tccagtttag aacctaattc ccaattcccg
gaccgggccc 60agccctgggc tcttactgtc cgcttttgct gggacctgtt ccacaaatgg
gcgtcttctg 120ccttgggccg tgggggttgg gccggaagct gcggacgcct
gggaaggggc cgctgcagct 180cttgagccgc ctctgcgggg accacttgca
ggccatccca gccaagaagg ccccggctgg 240gcaggaggag cctgggacgc
cgccctcctc gccgctgagt gccgagcagt tggaccggat 300ccagaggaac
aaggccgcgg ccctgctcag actcgcggcc cgcaacgtgc ccgtgggctt
360tggagagagc tggaagaagc acctcagcgg ggagttcggg aaaccgtatt
ttatcaagct 420aatgggattt gttgcagaag aaagaaagca ttacactgtt
tatccacccc cacaccaagt 480cttcacctgg acccagatgt gtgacataaa
agatgtgaag gttgtcatcc tgggacagga 540tccatatcat ggacctaatc
aagctcacgg gctctgcttt agtgttcaaa ggcctgttcc 600gcctccgccc
agtttggaga acatttataa agagttgtct acagacatag aggattttgt
660tcatcctggc catggagatt tatctgggtg ggccaagcaa ggtgttctcc
ttctcaacgc 720tgtcctcacg gttcgtgccc atcaagccaa ctctcataag
gagcgaggct gggagcagtt 780cactgatgca gttgtgtcct ggctaaatca
gaactcgaat ggccttgttt tcttgctctg 840gggctcttat gctcagaaga
agggcagtgc cattgatagg aagcggcacc atgtactaca 900gacggctcat
ccctcccctt tgtcagtgta tagagggttc tttggatgta gacacttttc
960aaagaccaat gagctgctgc agaagtctgg caagaagccc attgactgga
aggagctgtg 1020atcatcagct gaggggtggc ctttgagaag ctgctgttaa
cgtatttgcc agttacgaag 1080ttccactgaa aattttccta ttaattctta
agtactctgc ataaggggga aaagcttcca 1140gaaagcagcc atgaaccagg
ctgtccagga atggcagctg tatccaacca caaacaacaa 1200aggctaccct
ttgaccaaat gtctttctct gcaacatggc ttcggcctaa aatatgcaga
1260agacagatga ggtcaaatac tcagttggct ctctttatct cccttgcctt
tatggtgaaa 1320caggggagat gtgcaccttt caggcacagc cctagtttgg
cgcctgctgc tccttggttt 1380tgcctggtta gactttcagt gacagatgtt
ggggtgtttt tgcttagaaa ggtccccttg 1440tctcagcctt gcagggcagg
catgccagtc tctgccagtt ccactgcccc cttgatcttt 1500gaaggagtcc
tcaggcccct cgcagcataa ggatgttttg caactttcca gaatctggcc
1560cagaaattag ggctcaattt cctgattgta gtagaggtta agattgctgt
gagctttatc 1620agataagaga ccgagagaag taagctgggt cttgttattc
cttgggtgtt ggtggaataa 1680gcagtggaat ttgaacaagg aagaggagaa
aagggaattt tgtctttatg gggtggggtg 1740attttctcct agggttatgt
ccagttgggg tttttaaggc agcacagact gccaagtact 1800gtttttttta
accgactgaa atcactttgg gatatttttt cctgcaacac tggaaagttt
1860tagtttttta agaagtactc atgcagatat atatatatat atttttccca
gtcctttttt 1920taagagacgg tctttattgg gtctgcacct ccatccttga
tcttgttagc aatgctgttt 1980ttgctgttag tcgggttaga gttggctcta
cgcgaggttt gttaataaaa gtttgttaaa 2040agttcaaaaa aaaaaaaaaa aa
206241570DNAHomo sapiens 4aacgggatgg ggagctggac cagcagatta
tgagcttaca gaaagcctgg cctacatttt 60actctttttg gatttcttcc tcatcaagag
actgctgcag tgcctgtcat gtgacagcgg 120catggacata tgccccaggc
tttcctgctg gggtccatcc atgagcctgc aggtgccctc 180atggagcccc
agccctgccc tggaagcttg gctgagagct tcctggagga ggagcttcgg
240ctcaatgctg agctgagcca gctgcagttt tcggagcctg tgggcatcat
ctacaatccc 300gtggagtatg catgggagcc acatcgcaac tacgtgactc
gctactgcca gggccccaag 360gaagtactct tcctgggcat gaaccctgga
ccttttggca tggcccagac tggggtgccc 420tttggggaag taagcatggt
ccgggactgg ttgggcattg tggggcctgt gctgacccct 480ccccaagagc
atcctaaacg accagtgctg ggactggagt gcccacagtc agaagtgagt
540ggtgcccgat tctggggctt tttccggaac ctctgtggac agcctgaggt
cttcttccat 600cactgttttg tccacaatct atgccctctg cttttcctgg
ctcccagcgg gcgcaacctt 660actcctgctg agctgcctgc caagcagcga
gaacagcttc ttgggatctg tgatgcagcc 720ctctgccggc aggtgcagct
gctgggggtg cggctggtgg tgggagttgg gcgactggca 780gagcagcggg
cacgacgggc tctggcaggc ctgatgccag aggtccaggt ggaagggctc
840ctgcatccct ctccccgtaa cccacaggcc aacaagggct gggaggcagt
ggccaaggaa 900agattgaatg agctggggct gctgccactg ctgttgaaat
gagtgccctt ggggccttgc 960atgggacaca ttcaagacct cgaagtcatt
cttggccaag cagatgacaa cacatctcct 1020ggactggagc aaaaggtcct
tctgtgcacc ctggtcgctg ggaaacgtat tctttgatct 1080gttgaactgt
cttccaacct gccatggcag ttttgacact actcctgttt gccctcctga
1140ttcctgcttt ctttaccttt taacattgcc cctttcaggg gaccccactt
tgtagggaat 1200ctgcagaagg tgtgcttttg cacttgcaga ctgctctacc
tcagtgtttc cttgggagac 1260tttattcagc tgagagtgcc ctagacagta
acttctaagg tcacgtttac tatttcagag 1320gaaatatctt gccaggatac
ctacccatcc ttatagaaca gttaccttta gctgacccct 1380ttcctcacag
ggaccaagac aaagcatggg acatgaaatt aagagtgaac ttcttatggg
1440aggctgcagc tggatcagag gaaaaatcca gtgtgacaga gtgcaagtca
gaagacctgg 1500cttttcatcc cagctttgaa acttggaact ttttgattga
caaattaata aacctctcta 1560tgcctcaggc 157052470DNAHomo sapiens
5ggcggctgta gccgaggggg cggccggaaa gcagcggcgg cgtctggggc gctttcgcaa
60cattcagacc tcggttgcag cccggtgccg tgagctgaag aggtttcaca tcttactccg
120ccccacaccc tgggcgttgc ggcgctgggc tcgttgctgc agccggaccc
tgctcgatgg 180gcacgactgg gctggagagt ctgagtctgg gggaccgcgg
agctgccccc accgtcacct 240ctagtgagcg cctagtccca gacccgccga
atgacctccg caaagaagat gttgctatgg 300aattggaaag agtgggagaa
gatgaggaac aaatgatgat aaaaagaagc agtgaatgta 360atcccttgct
acaagaaccc atcgcttctg ctcagtttgg tgctactgca ggaacagaat
420gccgtaagtc tgtcccatgt ggatgggaaa gagttgtgaa gcaaaggtta
tttgggaaga 480cagcaggaag atttgatgtg tactttatca gcccacaagg
actgaagttc agatccaaaa 540gttcacttgc taattatctt cacaaaaatg
gagagacttc tcttaagcca gaagattttg 600attttactgt actttctaaa
aggggtatca agtcaagata taaagactgc agcatggcag 660ccctgacatc
ccatctacaa aaccaaagta acaattcaaa ctggaacctc aggacccgaa
720gcaagtgcaa aaaggatgtg tttatgccgc caagtagtag ttcagagttg
caggagagca 780gaggactctc taactttact tccactcatt tgcttttgaa
agaagatgag ggtgttgatg 840atgttaactt cagaaaggtt agaaagccca
aaggaaaggt gactattttg aaaggaatcc 900caattaagaa aactaaaaaa
ggatgtagga agagctgttc aggttttgtt caaagtgata 960gcaaaagaga
atctgtgtgt aataaagcag atgctgaaag tgaacctgtt gcacaaaaaa
1020gtcagcttga tagaactgtc tgcatttctg atgctggagc atgtggtgag
accctcagtg 1080tgaccagtga agaaaacagc cttgtaaaaa aaaaagaaag
atcattgagt tcaggatcaa 1140atttttgttc tgaacaaaaa acttctggca
tcataaacaa attttgttca gccaaagact 1200cagaacacaa cgagaagtat
gaggatacct ttttagaatc tgaagaaatc ggaacaaaag 1260tagaagttgt
ggaaaggaaa gaacatttgc atactgacat tttaaaacgt ggctctgaaa
1320tggacaacaa ctgctcacca accaggaaag acttcactgg tgagaaaata
tttcaagaag 1380ataccatccc acgaacacag atagaaagaa ggaaaacaag
cctgtatttt tccagcaaat 1440ataacaaaga agctcttagc cccccacgac
gtaaagcctt taagaaatgg acacctcctc 1500ggtcaccttt taatctcgtt
caagaaacac tttttcatga tccatggaag cttctcatcg 1560ctactatatt
tctcaatcgg acctcaggca aaatggcaat acctgtgctt tggaagtttc
1620tggagaagta tccttcagct gaggtagcaa gaaccgcaga ctggagagat
gtgtcagaac 1680ttcttaaacc tcttggtctc tacgatcttc gggcaaaaac
cattgtcaag ttctcagatg 1740aatacctgac aaagcagtgg aagtatccaa
ttgagcttca tgggattggt aaatatggca 1800acgactctta ccgaattttt
tgtgtcaatg agtggaagca ggtgcaccct gaagaccaca 1860aattaaataa
atatcatgac tggctttggg aaaatcatga aaaattaagt ctatcttaaa
1920ctctgcagct ttcaagctca tctgttatgc atagctttgc acttcaaaaa
agcttaatta 1980agtacaacca accacctttc cagccataga gattttaatt
agcccaacta gaagcctagt 2040gtgtgtgctt tcttaatgtg tgtgccaatg
gtggatcttt gctactgaat gtgtttgaac 2100atgttttgag atttttttaa
aataaattat tatttgacaa caatccaaaa aaaatacggc 2160ttttccaatg
atgaaatata atcagaagat gaaaaatagt tttaaactat caataataca
2220aagcaaattt ctatcagcct tgctaaagct aggggcccac taaatatttt
tatcggctag 2280gcgtggtggt gcatgcctgt aatctcggaa ggctgaggca
ggaggatcat ttgagctcat 2340gagggcccag gaggtcaagg cttcagtgag
ccatgatcat gccactgcac tccagtctgg 2400atgacagaga gagaccctgt
ctcaaaaaat atatatttaa aaaataaaaa taaaagctga 2460ccccaaagac
247063410DNAHomo sapiens 6gcaccaggcg cccagtggag ccgtttggga
gaattgcctg cgccacgcag cggggccgga 60caggcggtaa ggatctgatt aggctttcga
acttgagttt gactgatgtc ttctgtgtgg 120tgtccgctaa atcccacagc
atataggatc agtcgcattg gttataaggt ttgcttctgg 180ctgggtgcgg
tggctcatgc ctgtaatcca acattgggag gccaaggcag gcggaccacc
240tgaagtcggg agcttgagtc cagccactgt ctgggtactg ccagccatcg
ggcccaggtc 300tctggggttg tcttaccgca gtgagtacca cgcggtacta
cagagaccgg ctgcccgtgt 360gcccggcagg tggagccgcc gcatcagcgg
cctcggggaa tggaagcgga gaacgcgggc 420agctattccc ttcagcaagc
tcaagctttt tatacgtttc catttcaaca actgatggct 480gaagctccta
atatggcagt tgtgaatgaa cagcaaatgc cagaagaagt tccagcccca
540gctcctgctc aggaaccagt gcaagaggct ccaaaaggaa gaaaaagaaa
acccagaaca 600acagaaccaa aacaaccagt ggaacccaaa aaacctgttg
agtcaaaaaa atctggcaag 660tctgcaaaac caaaagaaaa acaagaaaaa
attacagaca catttaaagt aaaaagaaaa 720gtagaccgtt ttaatggtgt
ttcagaagct gaacttctga ccaagactct ccccgatatt 780ttgaccttca
atctggacat tgtcattatt ggcataaacc cgggactaat ggctgcttac
840aaagggcatc attaccctgg acctggaaac catttttgga agtgtttgtt
tatgtcaggg 900ctcagtgagg tccagctgaa ccatatggat gatcacactc
taccagggaa gtatggtatt 960ggatttacca acatggtgga aaggaccacg
cccggcagca aagatctctc cagtaaagaa 1020tttcgtgaag gaggacgtat
tctagtacag aaattacaga aatatcagcc acgaatagca 1080gtgtttaatg
gaaaatgtat ttatgaaatt tttagtaaag aagtttttgg agtaaaggtt
1140aagaacttgg aatttgggct tcagccccat aagattccag acacagaaac
tctctgctat 1200gttatgccat catccagtgc aagatgtgct cagtttcctc
gagcccaaga caaagttcat 1260tactacataa aactgaagga cttaagagat
cagttgaaag gcattgaacg aaatatggac 1320gttcaagagg tgcaatatac
atttgaccta cagcttgccc aagaggatgc aaagaagatg 1380gctgttaagg
aagaaaaata tgatccaggt tatgaggcag catatggtgg tgcttacgga
1440gaaaatccat gcagcagtga accttgtggc ttctcttcaa atgggctaat
tgagagcgtg 1500gagttaagag gagaatcagc tttcagtggc attcctaatg
ggcagtggat gacccagtca 1560tttacagacc aaattccttc ctttagtaat
cactgtggaa cacaagaaca ggaagaagaa 1620agccatgctt aagaatggtg
cttctcagct ctgcttaaat gctgcagttt taatgcagtt 1680gtcaacaagt
agaacctcag tttgctaact gaagtgtttt attagtattt tactctagtg
1740gtgtaattgt aatgtagaac agttgtgtgg tagtgtgaac cgtatgaacc
taagtagttt 1800ggaagaaaaa gtagggtttt tgtatactag cttttgtatt
tgaattaatt atcattccag 1860ctttttatat actatatttc atttatgaag
aaattgattt tcttttggga gtcactttta 1920atctgtaatt ttaaaataca
agtctgaata tttatagttg attcttaact gtgcataaac 1980ctagatatac
cattatccct tttataccta agaagggcat gctaataatt accactgtca
2040aagaggcaaa ggtgttgatt tttgtatata agttaagcct cagtggagtc
tcatttgtta 2100gtttttagtg gtaactaagg gtaaactcag ggttccctga
gctatatgca cactcagacc 2160tctttgcttt accagtggtg tttgtgagtt
gctcagtagt aaaaactggc ccttacctga 2220cagagccctg gctttgacct
gctcagccct gtgtgttaat cctctagtag ccaattaact 2280actctggggt
ggcaggttcc agagaatcga gtagaccttt tgccactcat ctgtgtttta
2340cttgagacat gtaaatatga tagggaagga actgaatttc tccattcata
tttataacca 2400ttctagtttt atcttccttg gctttaagag tgtgccatgg
aaagtgataa gaaatgaact 2460tctaggctaa gcaaaaagat gctggagata
tttgatactc tcatttaaac tggtgcttta 2520tgtacatgag atgtactaaa
ataagtaata tagaattttt cttgctaggt aaatccagta 2580agccaataat
tttaaagatt ctttatctgc atcattgctg tttgttacta taaattaaat
2640gaacctcatg gaaaggttga ggtgtatacc tttgtgattt tctaatgagt
tttccatggt 2700gctacaaata atccagacta ccaggtctgg tagatattaa
agctgggtac taagaaatgt 2760tatttgcatc ctctcagtta ctcctgaata
ttctgatttc atacgtaccc agggagcatg 2820ctgttttgtc aatcaatata
aaatatttat gaggtctccc ccacccccag gaggttatat 2880gattgctctt
ctctttataa taagagaaac aaattcttat tgtgaatctt aacatgcttt
2940ttagctgtgg ctatgatgga ttttattttt tcctaggtca agctgtgtaa
aagtcattta 3000tgttatttaa atgatgtact gtactgctgt ttacatggac
gttttgtgcg ggtgctttga 3060agtgccttgc atcagggatt aggagcaatt
aaattatttt ttcacgggac tgtgtaaagc 3120atgtaactag gtattgcttt
ggtatataac tattgtagct ttacaagaga ttgttttatt 3180tgaatgggga
aaataccctt taaattatga cggacatcca ctagagatgg gtttgaggat
3240tttccaagcg tgtaataatg atgtttttcc taacatgaca gatgagtagt
aaatgttgat 3300atatcctata catgacagtg tgagactttt tcattaaata
atattgaaag attttaaaat 3360tcatttgaaa gtctgatggc ttttacaata
aaagatatta agaattgtta 341071108DNAHomo sapiens 7cctgggcccc
catgcccgtg cagctcgcac atatgtgggg cagagcagcc accctgcccc 60cagcagcagc
cgtccatcgt cagacgtgat catttcctga ggcctcgagt gtgtcagggt
120gtttgtgcct cataacaacc cacaggatgg tcacccccgc tttgcagatg
aagaaaccaa 180agcagttttg ccgacggatg gggcaaaaga agcagcgacc
agctagagca gggcagccac 240acagctcgtc cgacgcagcc caggcacctg
cagagcagcc acacagctcg tccgatgcag 300cccaggcacc ttgccccagg
gagcgctgct tgggaccgcc caccactccg ggcccatacc 360gcagcatcta
tttctcaagc ccaaagggcc accttacccg actggggttg gagttcttcg
420accagccggc agtccccctg gcccgggcat ttctgggaca ggtcctagtc
cggcgacttc 480ctaatggcac agaactccga ggccgcatcg tggagaccga
ggcatacctg gggccagagg 540atgaaccggc ccactcaagg ggtggccggc
agaccccccg caaccgaggc atgttcatga 600agccggggac cctgtacgtg
tacatcattt acggcatgta cttctgcatg aacatctcca 660gccaggggga
cggggcttgc gtcttgctgc gagcactgga gcccctggaa ggtctggaga
720ccatgcgtca cgttcgcagc accctccgga aaggcaccgc cagccgtgtc
ctcaaggacc 780gcgagctctg cagtggcccc tccaagctgt gccaggccct
ggccatcaac aagagctttg 840accagaggga cctggcacag gatgaagctg
tatggctgga gcgtggtccc ctggagccca 900gtgagccggc tgtagtggca
gcagcccggg tgggcgtcgg ccatgcaggg gagtgggccc 960ggaaacccct
ccgcttctat gtccggggca gcccctgggt cagtgtggtc gacagagtgg
1020ctgagcagga cacacaggcc tgagcaaagg gcctgcccag acaagatttt
ttaattgttt 1080aaaaaccgaa taaatgtttt atttctag 11088939DNAHomo
sapiens 8atgtgtagtc cgcaggagtc cggcatgacc gccttgagcg cgaggatgct
gacccggagc 60cggagcctgg gacccggggc tgggccgcgg gggtgtaggg aggagcccgg
gcctctccgg 120agaagagagg ctgcagcaga agcgaggaaa agccacagcc
ccgtgaagcg tccgcggaaa 180gcacagagac tgcgtgtggc ctatgagggc
tcggacagtg agaaaggtga gggggctgag 240cccctcaagg tgccagtctg
ggagccccag gactggcagc aacagctggt caacatccgt 300gccatgagga
acaaaaagga tgcacctgtg gaccatctgg ggactgagca ctgctatgac
360tccagtgccc ccccaaaggt acgcaggtac caggtgctgc tgtcactgat
gctctccagc 420caaaccaaag accaggtgac ggcgggcgcc atgcagcgac
tgcgggcgcg gggcctgacg 480gtggacagca tcctgcagac agatgatgcc
acgctgggca agctcatcta ccccgtcggt 540ttctggagga gcaaggtgaa
atacatcaag cagaccagcg ccatcctgca gcagcactac 600ggtggggaca
tcccagcctc tgtggccgag ctggtggcgc tgccgggtgt tgggcccaag
660atggcacacc tggctatggc tgtggcctgg ggcactgtgt caggcattgc
agtggacacg 720catgtgcaca gaatcgccaa caggctgagg tggaccaaga
aggcaaccaa gtccccagag 780gagacccgcg ccgccctgga ggagtggctg
cctagggagc tgtggcacga gatcaatgga 840ctcttggtgg gcttcggcca
gcagacctgt ctgcctgtgc accctcgctg ccacgcctgc 900ctcaaccaag
ccctctgccc ggccgcccag ggtctctga 93991854DNAHomo sapiens 9ggagcctcta
gaactatgag cccgaggcct tcccctctcc cagagcgcag aggctttgaa 60ggctacctct
gggaagccgc tcaccgtcgg aagctgcggg agctgaaact gcgccatcgt
120cactgtcggc ggccatgaca ccgctcgtct cccgcctgag tcgtctgtgg
gccatcatga 180ggaagccacg agcagccgtg ggaagtggtc acaggaagca
ggcagccagc caggaaggga 240ggcagaagca tgctaagaac aacagtcagg
ccaagccttc tgcctgtgat gggatgattg 300ctgagtgtcc tggggcccca
gcaggcctgg ccaggcagcc ggaagaggtg gtattgcagg 360cctctgtctc
ctcataccat ctattcagag acgtagctga agtcacagcc ttccgaggga
420gcctgctaag ctggtacgac caagagaaac gggacctacc atggagaaga
cgggcagaag 480atgagatgga cctggacagg cgggcatatg ctgtgtgggt
ctcagaggtc atgctgcagc 540agacccaggt tgccactgtg atcaactact
ataccggatg gatgcagaag tggcctacac 600tgcaggacct ggccagtgct
tccctggagg aggtgaatca actctgggct ggcctgggct 660actattctcg
tggccggcgg ctgcaggagg gagctcggaa ggtggtagag gagctagggg
720gccacatgcc acgtacagca gagaccctgc agcagctcct gcctggcgtg
gggcgctaca 780cagctggggc cattgcctct atcgcctttg gccaggcaac
cggtgtggtg gatggcaacg 840tagcacgggt gctgtgccgt gtccgagcca
ttggtgctga tcccagcagc acccttgttt 900cccagcagct ctggggtcta
gcccagcagc tggtggaccc agcccggcca ggagatttca 960accaagcagc
catggagcta ggggccacag tgtgtacccc acagcgccca ctgtgcagcc
1020agtgccctgt ggagagcctg tgccgggcac gccagagagt ggagcaggaa
cagctcttag 1080cctcagggag cctgtcgggc agtcctgacg tggaggagtg
tgctcccaac actggacagt 1140gccacctgtg cctgcctccc tcggagccct
gggaccagac cctgggagtg gtcaacttcc 1200ccagaaaggc cagccgcaag
ccccccaggg aggagagctc tgccacctgt gttctggaac 1260agcctggggc
ccttggggcc caaattctgc tggtgcagag gcccaactca ggtctgctgg
1320caggactgtg ggagttcccg tccgtgacct gggagccctc agagcagctt
cagcgcaagg 1380ccctgctgca ggaactacag cgttgggctg ggcccctccc
agccacgcac ctccggcacc 1440ttggggaggt tgtccacacc ttctctcaca
tcaagctgac atatcaagta tatgggctgg 1500ccttggaagg gcagacccca
gtgaccaccg taccaccagg tgctcgctgg ctgacgcagg 1560aggaatttca
caccgcagct gtttccaccg ccatgaaaaa ggttttccgt gtgtatcagg
1620gccaacagcc agggacctgt atgggttcca aaaggtccca ggtgtcctct
ccgtgcagtc 1680ggaaaaagcc ccgcatgggc cagcaagtcc tggataattt
ctttcggtct cacatctcca 1740ctgatgcaca cagcctcaac agtgcagccc
agtgacacct ctgaaagccc ccattccctg 1800agaatcctgt tgttagtaaa
gtgcttattt ttgtagttaa aaaaaaaaaa aaaa 1854102557DNAHomo
sapiensmisc_feature(200)..(200)any nucleotide 10ttcgcttgaa
cccgggaggc ggagcttgca gtgagccgag atcgcgccat cacactccag 60ctcaggcgac
agagtgagac tccgtctcaa agaaaaaaaa cttgcagcct gatagttaag
120atacagcaac cccaaatccc tatgctaaaa ggtgagaatg gcccagataa
aggtcatgtc 180tcctagctcc ctgctttttn atgccatcct ccagaaggga
agaaattaaa taatccatcc 240tcctactcca ggcgactaga aggcaggctg
cctcagggcc acacactggg acttggactc 300aacctgatgg gcttctgggc
ccagccccag acaaaccccc ggcaaacgtc ccattccgag 360gaaagcatga
gcagatggag tatggaagaa atgcccaaga cggcaggcag cagctgtggc
420ggccggcggg acgacaatcc gaggagaggc ctctgatgtc ctgaggtctc
agaggacgcc 480taaaggcctt gaatgggaca agcttagcgg gcgggcgcag
aagagaataa tactctggag 540acacttcccg agggctctgg ggccggagct
gtgttcgctc cggttcttgg tgaagacagg 600gttcgtggga ggcggcccaa
ggagggcgaa cgcctaagac tgcaaaggct cgggggagaa 660cggctctcgg
agaacgggct ggggaaggac gtggctctga agacggacag ccctgaggaa
720ccgcggggcg cccagatgga actcgttagc gccccgagtg cagacaatcc
cggaggggga 780aaggcgagca gctggcagag agcccagtgc cggccaaccg
cgcgagcgcc tcagaacggc 840ccgcccaccc tgatttctca ttggcgcctc
ctacctcctc ctcggattgg ctacctctag 900gtgaaatgag cggtggttga
gccctacttc cggtggtgct gtggtctgcc cctggagaac 960ccagaagaac
acagctgtgc gcgcccacag gctctggggg cgggagaaga
taagtcgcaa 1020ggagggggcg ggacctacac ctcaggaaag ccggagaatt
ggggcacgaa gcggggcttt 1080gatgacccgc aaagggcgag gcatgcagga
ggtggaggaa ttaagtgaaa cagggaaggt 1140tgttaaacag caccgtgtgg
gcgaggcctt aagggtcgtg gtccttgtct gggcggggtc 1200tttgggcgtc
gacgaggcct ggttctgggt aggcggggct actacggggc ggtgcctgct
1260gtggaaatgc ctgcccgcgc gcttctgccc aggcgcatgg ggcatcgtac
tctagcctcc 1320actcctgccc tgtgggcctc catcccgtgc cctcgctctg
agctgcgcct ggacctggtt 1380ctgccttctg gacaatcttt ccggtggagg
gagcaaagtc ctgcacactg gagtggtgta 1440ctagcggatc aagtatggac
actgactcag actgaggagc agctccactg cactgtgtac 1500cgaggagaca
agagccaggc tagcaggccc acaccagacg agctggaggc cgtgcgcaag
1560tacttccagc tagatgttac cctggctcaa ctgtatcacc actggggttc
cgtggactcc 1620cacttccaag aggtggctca gaaattccaa ggtgtgcgac
tgctgcgaca agaccccatc 1680gaatgccttt tctcttttat ctgttcctcc
aacaacaaca tcgcccgcat cactggcatg 1740gtggagcggc tgtgccaggc
ttttggacct cggctcatcc agcttgatga tgtcacctac 1800catggcttcc
ccagcctgca ggccctggct gggccagagg tggaggctca tctcaggaag
1860ctgggcctgg gctatcgtgc ccgttacgtg agtgccagtg cccgagccat
cctggaagaa 1920cagggcgggc tagcctggct gcagcagcta cgagagtcct
catatgagga ggcccacaag 1980gccctctgca tcctgcctgg agtgggcacc
aaggtggctg actgcatctg cctgatggcc 2040ctagacaagc cccaggctgt
gcccgtggat gtccatatgt ggcacattgc ccaacgtgac 2100tacagctggc
accctaccac gtcccaggcg aagggaccga gcccccagac caacaaggaa
2160ctgggaaact ttttccggag cctgtgggga ccttatgctg gctgggccca
agcggtgctg 2220ttcagtgccg acctgcgcca atcccgccat gctcaggagc
caccagcaaa gcgcagaaag 2280ggttccaaag ggccggaagg ctagatgggg
caccctggac aaagaaattc cccaagcacc 2340ttcccctcca ttccccactt
ctctctcccc atccccaccc agtctcatgt tggggagggg 2400cctccctgtg
actacctcaa aggccaggca cccccaaatc aagcagtcag tttgcacaac
2460aagatggggt gggggatatt gagggagaca gcgctaagga tggttttatc
ttccctttat 2520tacaagaagg aacaataaaa tagaaacatt tgtatgg
255711643DNAHomo sapiens 11gagcggcggt gcagaaccca gggaccatgg
gcgcctccag gctctatacc ctggtgctgg 60tcctgcagcc tcagcgagtt ctcctgggca
tgaaaaagcg aggcttcggg gccggccggt 120ggaatggctt tgggggcaaa
gtgcaagaag gagagaccat cgaggatggg gctaggaggg 180agctgcagga
ggagagcggt ctgacagtgg acgccctgca caaggtgggc cagatcgtgt
240ttgagttcgt gggcgagcct gagctcatgg acgtgcatgt cttctgcaca
gacagcatcc 300aggggacccc cgtggagagc gacgaaatgc gcccatgctg
gttccagctg gatcagatcc 360ccttcaagga catgtggccc gacgacagct
actggtttcc actcctgctt cagaagaaga 420aattccacgg gtacttcaag
ttccagggtc aggacaccat cctggactac acactccgcg 480aggtggacac
ggtctagcgg gagcccaggg cagcccctgg gcaggagacg tggctgctga
540acagctgcaa accatcttca cctgggggca ttgagtggcg cagagccggg
tttcatctgg 600aattaactgg atggaaggga aaataaagct atctagcggt gaa
643121006DNAHomo sapiens 12cgtctcctcg ctcgccttct ggctctgcca
tgccctgctc tgaagagaca cccgccattt 60cacccagtaa gcgggcccgg cctgcggagg
tgggcggcat gcagctccgc tttgcccggc 120tctccgagca cgccacggcc
cccacccggg gctccgcgcg cgccgcgggc tacgacctgt 180acagtgccta
tgattacaca ataccaccta tggagaaagc tgttgtgaaa acggacattc
240agatagcgct cccttctggg tgttatggaa gagtggctcc acggtcaggc
ttggctgcaa 300aacactttat tgatgtagga gctggtgtca tagatgaaga
ttatagagga aatgttggtg 360ttgtactgtt taattttggc aaagaaaagt
ttgaagtcaa aaaaggtgat cgaattgcac 420agctcatttg cgaacggatt
ttttatccag aaatagaaga agttcaagcc ttggatgaca 480ccgaaagggg
ttcaggaggt tttggttcca ctggaaagaa ttaaaattta tgccaagaac
540agaaaacaag aagtcatacc tttttcttaa aaaaaaaaaa agtttttgct
tcaagtgttt 600tggtgttttg cacttctgta aacttactag ctttaccttc
taaaagtact gcatttttta 660ctttttttta tgatcaagga aaagatcatt
aaaaaaaaac acaaaagaag tttttctttg 720tgtttggatc aaaaagaaac
tttgtttttc cgcaattgaa ggttgtatgt aaatctgctt 780tgtggtgacc
tgatgtaaac agtgtcttct taaaatcaaa tgtaaatcaa ttacagatta
840aaaaaaaaaa gcctgtattt aactcatatg atctcccttc agcaacttat
tttgctttaa 900ttgctttaaa tcttaagcaa tattttttat tcagtaaaca
aattctttca caaggtacaa 960aatcttgcat aagctgaact aaaataaaaa
tgaaaaggag agatta 1006131420DNAHomo sapiens 13tgccatcggg ccggtgcaga
tacggggttg ctcttttgct cataagaggg gcttcgctgg 60cagtctgaac ggcaagcttg
agtcaggacc cttaattaag atcctcaatt ggctggaggg 120cagatctcgc
gagtagggca acgcggtaaa aatattgctt cggtgggtga cgcggtacag
180ctgcccaagg gcgttcgtaa cgggaatgcc gaagcgtggg aaaaagggag
cggtggcgga 240agacggggat gagctcagga cagagccaga ggccaagaag
agtaagacgg ccgcaaagaa 300aaatgacaaa gaggcagcag gagagggccc
agccctgtat gaggaccccc cagatcacaa 360aacctcaccc agtggcaaac
ctgccacact caagatctgc tcttggaatg tggatgggct 420tcgagcctgg
attaagaaga aaggattaga ttgggtaaag gaagaagccc cagatatact
480gtgccttcaa gagaccaaat gttcagagaa caaactacca gctgaacttc
aggagctgcc 540tggactctct catcaatact ggtcagctcc ttcggacaag
gaagggtaca gtggcgtggg 600cctgctttcc cgccagtgcc cactcaaagt
ttcttacggc ataggcgatg aggagcatga 660tcaggaaggc cgggtgattg
tggctgaatt tgactcgttt gtgctggtaa cagcatatgt 720acctaatgca
ggccgaggtc tggtacgact ggagtaccgg cagcgctggg atgaagcctt
780tcgcaagttc ctgaagggcc tggcttcccg aaagcccctt gtgctgtgtg
gagacctcaa 840tgtggcacat gaagaaattg accttcgcaa ccccaagggg
aacaaaaaga atgctggctt 900cacgccacaa gagcgccaag gcttcgggga
attactgcag gctgtgccac tggctgacag 960ctttaggcac ctctacccca
acacacccta tgcctacacc ttttggactt atatgatgaa 1020tgctcgatcc
aagaatgttg gttggcgcct tgattacttt ttgttgtccc actctctgtt
1080acctgcattg tgtgacagca agatccgttc caaggccctc ggcagtgatc
actgtcctat 1140caccctatac ctagcactgt gacaccaccc ctaaatcact
ttgagcctgg gaaataagcc 1200ccctcaacta ccattccttc tttaaacact
cttcagagaa atctgcattc tatttctcat 1260gtataaaact aggaatcctc
caaccaggct cctgtgatag agttctttta agcccaagat 1320tttttatttg
agggtttttt gttttttaaa aaaaaattga acaaagacta ctaatgactt
1380tgtttgaatt atccacatga aaataaagag ccatagtttc 1420141259DNAHomo
sapiens 14ccggagctgg gttgctcctg ctcccgtctc caagtcctgg tacctccttc
aagctgggag 60agggctctag tccctggttc tgaacactct ggggttctcg ggtgcaggcc
gccatgagca 120aacggaaggc gccgcaggag actctcaacg ggggaatcac
cgacatgctc acagaactcg 180caaactttga gaagaacgtg agccaagcta
tccacaagta caatgcttac agaaaagcag 240catctgttat agcaaaatac
ccacacaaaa taaagagtgg agctgaagct aagaaattgc 300ctggagtagg
aacaaaaatt gctgaaaaga ttgatgagtt tttagcaact ggaaaattac
360gtaaactgga aaagattcgg caggatgata cgagttcatc catcaatttc
ctgactcgag 420ttagtggcat tggtccatct gctgcaagga agtttgtaga
tgaaggaatt aaaacactag 480aagatctcag aaaaaatgaa gataaattga
accatcatca gcgaattggg ctgaaatatt 540ttggggactt tgaaaaaaga
attcctcgtg aagagatgtt acaaatgcaa gatattgtac 600taaatgaagt
taaaaaagtg gattctgaat acattgctac agtctgtggc agtttcagaa
660gaggtgcaga gtccagtggt gacatggatg ttctcctgac ccatcccagc
ttcacttcag 720aatcaaccaa acagccaaaa ctgttacatc aggttgtgga
gcagttacaa aaggttcatt 780ttatcacaga taccctgtca aagggtgaga
caaagttcat gggtgtttgc cagcttccca 840gtaaaaatga tgaaaaagaa
tatccacaca gaagaattga tatcaggttg atacccaaag 900atcagtatta
ctgtggtgtt ctctatttca ctgggagtga tattttcaat aagaatatga
960gggctcatgc cctagaaaag ggtttcacaa tcaatgagta caccatccgt
cccttgggag 1020tcactggagt tgcaggagaa cccctgccag tggatagtga
aaaagacatc tttgattaca 1080tccagtggaa ataccgggaa cccaaggacc
ggagcgaatg aggcctgtat cctccctggc 1140agacacaacc caataggagt
cttaatttat ttcttaacct ttgctatgta agggtctttg 1200gtgtttttaa
atgattgttt cttcttcatg cttttgcttg caatgtagtc aataaaacc 1259
* * * * *