U.S. patent application number 10/240589 was filed with the patent office on 2004-04-22 for diagnosis of diseases associated with dna repair.
Invention is credited to Berlin, Kurt, Olek, Alexander, Piepenbrock, Christian.
Application Number | 20040076956 10/240589 |
Document ID | / |
Family ID | 27437807 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040076956 |
Kind Code |
A1 |
Olek, Alexander ; et
al. |
April 22, 2004 |
Diagnosis of diseases associated with dna repair
Abstract
Chemically modified genomic sequences of genes associated with
DNA repair, to oligonucleotides and/or PNA-oligomers for detecting
the cytosine methylation state of genes associated with DNA repair
which are directed against the sequence are disclosed. In addition,
a method for ascertaining genetic and/or epigenetic parameters of
genes associated with DNA repair is disclosed.
Inventors: |
Olek, Alexander; (Berlin,
DE) ; Piepenbrock, Christian; (Berlin, DE) ;
Berlin, Kurt; (Stahnsdorf, DE) |
Correspondence
Address: |
Davidson Davidson & Kappel
485 Senventh Avenue 14th Floor
New York
NY
10018
US
|
Family ID: |
27437807 |
Appl. No.: |
10/240589 |
Filed: |
March 10, 2003 |
PCT Filed: |
April 6, 2001 |
PCT NO: |
PCT/EP01/03972 |
Current U.S.
Class: |
435/6.12 ;
536/24.3 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
29/00 20180101; C12Q 2600/156 20130101; C12Q 1/6883 20130101; A61P
13/12 20180101; C12Q 2600/154 20130101; C12Q 1/6886 20130101; A61P
35/00 20180101; C07K 14/82 20130101; A61P 7/04 20180101; A61P 11/06
20180101; C07K 14/4703 20130101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 6, 2000 |
DE |
10019058.8 |
Apr 7, 2000 |
DE |
10019173.8 |
Jun 30, 2000 |
DE |
10032529.7 |
Sep 1, 2000 |
DE |
10043826.1 |
Claims
1. A nucleic acid comprising a sequence at least 18 bases in length
of a segment of the chemically pretreated DNA of genes associated
with DNA repair according to one of the sequences taken from the
group of Seq. ID No.1 to Seq. ID No.144 and sequences complementary
thereto.
2. A nucleic acid comprising a sequence at least 18 base pairs in
length of a segment of the chemically pretreated DNA of genes
associated with DNA repair according to a sequence according to one
of the genes PMS2L1 (D38435), PMS2L12 (AF053356), PMS2L2 (D38436),
PMS2L3 (D38437), PMS2L4 (D38438 and D38500), PMS2L5 (D38439),
PMS2L6 (D38440), MGMT (NM.sub.--002412), MSH2 (NM.sub.--000251),
NUDTI (NM.sub.--002452), TDG (NM.sub.--003211), INPPL1
(NM.sub.--001567), RFC4 (NM.sub.--002916), DDIT1L, FANCB, XRCC8 and
sequences complementary thereto:
3. An oligomer, in particular an oligonucleotide or peptide nucleic
acid (PNA)-oligomer, said oligomer comprising in each case at least
one base sequence having a length of at least 9 nucleotides which
hybridizes to or is identical to a chemically pretreated DNA of
genes associated with DNA repair according to one of the Seq ID Nos
1 to 144 according to claim 1 or to a chemically pretreated DNA of
genes according to claim 2 and sequences complementary thereto.
4. The oligomer as recited in claim 3; wherein the base sequence
includes at least one CpG dinucleotide.
5. The oligomer as recited in claim 3; characterized in that the
cytosine of the CpG dinucleotide is located approximately in the
middle third of the oligomer.
6. A set of oligomers, comprising at least two oligomers according
to any of claims 3 to 5.
7. A set of oligomers as recited in claim 6, comprising oligomers
for detecting the methylation state of all CpG dinucleotides within
one of the sequences according to Seq. ID Nos. 1 through 144
according to claim 1 or a chemically pretreated DNA of genes
according to claim 2, and sequences complementary thereto
8. A set of at least two oligonucleotides as recited in claim 3,
which can be used as primer oligonucleotides for the amplification
of DNA sequences of one of Seq. ID No. 1 through Seq. ID No. 144
and sequences complementary thereto and/or sequences of a
chemically pretreated DNA of genes according to claim 2, and
sequences complementary thereto and segments thereof.
9. A set of oligonucleotides as recited in claim 8, characterized
in that at least one oligonucleotide is bound to a solid phase.
10. Use of a set of oligomer probes comprising at least ten of the
oligomers according to any of claims 6 through 9 for detecting the
cytosine methylation state and/or single nucleotide polymorphisms
(SNPS) in a chemically pretreated genomic DNA according to claim 1
or a chemically pretreated DNA of genes according to claim 2.
11. A method for manufacturing an arrangement of different
oligomers (array) fixed to a carrier material for analyzing
diseases associated with the methylation state of the CpG
dinucleotides of one of the Seq. ID No. 1 through Seq. ID No. 144
and sequences complementary thereto and/or chemically pretreated
DNA of genes according to claim 2, wherein at least one oligomer
according to any of the claims 3 through 5 is coupled to a solid
phase.
12. An arrangement of different oligomers (array) obtainable
according to claim 11.
13. An array of different oligonucleotide- and/or PNA-oligomer
sequences as recited in claim 12, characterized in that these are
arranged on a plane solid phase in the form of a rectangular or
hexagonal lattice.
14. The array as recited in any of the claims 12 or 13,
characterized in that the solid phase surface is composed of
silicon, glass, polystyrene, aluminium, steel, iron, copper,
nickel, silver, or gold.
15. A DNA- and/or PNA-array for analyzing diseases associated with
the methylation state of genes, comprising at least one nucleic
acid according to one of the preceeding claims.
16. A method for ascertaining genetic and/or epigenetic parameters
for the diagnosis and/or therapy of existing diseases or the
predisposition to specific diseases by analyzing cytosine
methylations, characterized in that the following steps are carried
out: a) in a genomic DNA sample, cytosine bases which are
unmethylated at the 5-position are converted, by chemical
treatment, to uracil or another base which is dissimilar to
cytosine in terms of hybridization behavior; b) fragments of the
chemically pretreated genomic DNA are amplified using sets of
primer oligonucleotides according to claim 8 or 9 and a polymerase,
the amplificates carrying a detectable label; c) Amplificates are
hybridized to a set of oligonucleotides and/or PNA probes according
to the claims 6 and 7, or else to an array according to one of the
claims 12 through 15; d) the hybridized amplificates are
subsequently detected.
17. The method as recited in claim 16, characterized in that the
chemical treatment is carried out by means of a solution of a
bisulfite, hydrogen sulfite or disulfite.
18. The method as recited in one of the claims 16 or 17,
characterized in that more than ten different fragments having a
length of 100-2000 base pairs are amplified.
19. The method as recited in one of the claims 16 through 18,
characterized in that the amplification of several DNA segments is
carried out in one reaction vessel.
20. The method as recited in one of the claims 16 through 19,
characterized in that the polymerase is a heat-resistant DNA
polymerase.
21. The method as recited in claim 20, characterized in that the
amplification is carried out by means of the polymerase chain
reaction (PCR).
22. The method as recited in one of the claims 16 through 21,
characterized in that the labels of the amplificates are
fluorescence labels.
23. The method as recited in one of the claims 16 through 21,
characterized in that the labels of the amplificates are
radionuclides.
24. The method as recited in one of the claims 16 through 21,
characterized in that the labels of the amplificates are detachable
molecule fragments having a typical mass which are detected in a
mass spectrometer.
25. The method as recited in one of the claims 16 through 21,
characterized in that the amplificates or fragments of the
amplificates are detected in the mass spectrometer.
26. The method as recited in one of the claims 24 and/or 25,
characterized in that the produced fragments have a single positive
or negative net charge for better detectability in the mass
spectrometer.
27. The method as recited in one of the claims 24 through 26,
characterized in that detection is carried out and visualized by
means of matrix assisted laser desorption/ionization mass
spectrometry (MALDI) or using electron spray mass spectrometry
(ESI).
28. The method as recited in one of the claims 16 through 27,
characterized in that the genomic DNA is obtained from cells or
cellular components-which contain DNA, sources of DNA comprising,
for example, cell lines, biopsies, blood, sputum, stool, urine,
cerebral-spinal fluid, tissue embedded in paraffin such as tissue
from eyes, intestine, kidney, brain, heart, prostate, lung, breast
or liver, histologic object slides, and all possible combinations
thereof.
29. A kit comprising a bisulfite (=disulfite, hydrogen sulfite)
reagent as well as oligonucleotides and/or PNA-oligomers according
to one of the claims 3 through 5.
30. The use of a nucleic acid according to claims 1 or 2, of an
oligonucleotide or PNA-oligomer according to one of the claims 3
through 5, of a kit according to claim 29, of an array according to
one of the claims 12 through 15, of a set of oligonucleotides
according to one of claims 6 through 9 for the diagnosis of Ataxia
telangiectasia, Ageing, Bloom's Syndrome, Immunodeficiency,
Cockayne syndrome, Nijmegen breakage syndrome, Trichothiodystrophy,
Fanconi Anaemia, Werner Syndrome, solid tumors and cancer.
31. The use of a nucleic acid according to claims 1 or 2, of an
oligonucleotide or PNA-oligomer according to one of claims 3
through 5, of a kit according to claim 29, of an array according to
one of the claims 12 through 15, of a set of oligonucleotides
according to one of claims 6 through 9 for the therapy of Ataxia
telangiectasia, Ageing, Bloom's Syndrome, Immunodeficiency,
Cockayne syndrome, Nijmegen breakage syndrome, Trichothiodystrophy,
Fanconi Anaemia, Werner Syndrome, solid tumors and cancer.
32. A kit, comprising a bisulfite (=disulfite, hydrogen sulfite)
reagent as well as oligonucleotides and/or PNA-oligomers according
to one of claims 3 through 5.
Description
[0001] The levels of observation that have been well studied by the
methodological developments of recent years in molecular biology,
are the genes themselves, the translation of these genes into RNA,
and the resulting proteins. The question of which gene is switched
on at which point in the course of the development of an
individual, and how the activation and inhibition of specific genes
in specific cells and tissues are controlled is correlatable to the
degree and character of the methylation of the genes or of the
genome. In this respect, pathogenic conditions may manifest
themselves in a changed methylation pattern of individual genes or
of the genome.
[0002] The present invention relates to nucleic acids,
oligonucleotides, PNA-oligomers and to a method for the diagnosis
and/or therapy of diseases which have a connection with the genetic
and/or epigenetic parameters of genes associated with DNA repair
and, in particular, with the methylation status thereof.
PRIOR ART
[0003] The ability to repair DNA damage is an essential component
of the genetic mechanisms conserving genomic fidelity. DNA damage
may take several forms, including single- and double-strand breaks,
inter-and intrastrand crosslinks and different kinds of base
modifications. DNA damage may be the result of a variety of
factors. Common exogenous sources of DNA damage include, chemical
compounds and irradiation. Endogenous sources include spontaneous
chemical conversion (e.g. deamination or depurination), the effect
of oxygen and free radicals (causing base damage and DNA strand
breaks), and malfunctions in DNA replication mechanisms (causing
base mismatches and deletions). At the cellular level DNA damage
may affect functions such as transcription, DNA replication, cell
cycle, apoptosis and mutagenesis. At the phenotypic level this can
lead to the development of diseases such as cancer and ageing. Each
cell has several complex methods in place to deal with both single
base, or structural mismatches. Common repair pathways for double
stranded breaks are homologous recombination based mechanisms.
Another common mechanism for double stranded DNA break repair is
non-homologous end joining. The mechanisms of double stranded break
repair, and the diseases associated with them have been reviewed by
Khanna and Jackson "DNA double-strand breaks: signalling, repair
and the cancer connection." Nature Genetics March 2001
;27(3):247-254.
[0004] Small base mismatches are generally removed by base excision
repair mechanisms, these comprise specific glycosylases that remove
bases, followed by polymerases and ligases that fill in the gap
left by the excision. Nucleotide excision repair (NER) removes a
wide diversity of lesions, which include UV-induced lesions, bulky
chemical adducts, several forms of base mismatches and some forms
of oxidative damage. Several variations on the pathway exist, such
as global genome nucleotide excision repair and
transcription-coupled nucleotide excision repair. The generalised
NER process involves the action of at least 30 proteins in a
mechanism involving damage identification, localised unwinding of
the DNA helix, excision of the damaged portion of DNA, synthesis of
a new strand and subsequent ligation. The consequences of a defect
in one of the NER proteins are apparent from three rare recessive
photosensitive syndromes: xeroderma pigmentosum (XP), CS and the
photosensitive form of the brittle hair disorder
trichothiodystrophy (TTD), see below for further references. A
further overview of DNA repair mechanisms is available from
standard molecular biology textbooks such as Alberts et. al.
`Molecular biology of the cell` Garland Publishing.
[0005] That these mechanisms are highly conserved between species
highlights their importance. Malfunctions in DNA repair pathways
have been implicated in a number of diseases, including cancer and
ageing. Diseases associated with DNA repair mechanisms include the
following:
[0006] Ataxia telangiectasia: Allen et. al. `Ataxia telangiectasia
mutated is essential during adult neurogenesis.` Genes and
Development Mar. 1, 2001;15(5):554-566.
[0007] Ageing: Martin et. al. `Genetic analysis of ageing: role of
oxidative damage and environmental stresses` Nature Genetics May
1996; 13 (1): 25.
[0008] Bloom's Syndrome: Karow et. al. `The Bloom's syndrome gene
product promotes branch migration of holliday junctions.` Proc Natl
Acad Sci U S A Jun. 6, 2000;97(12):6504-8.
[0009] Immunodeficiency: Gennery et. al. `Immunodeficiency
associated with DNA repair defects.` Clin Exp Immunol July
2000;121(1):1-7.
[0010] Cockayne syndrome: Hanawalt `DNA repair: The bases for
Cockayne syndrome` Nature 405,(2000): 415-416.
[0011] Nijmegen breakage syndrome: Digweed et. al. `Nijmegen
breakage syndrome: consequences of defective DNA double strand
break repair.` Bioessays August 1999;21(8):649-56.
[0012] Trichothiodystrophy:Vermeulen et. al. `Sublimiting
concentration of TFIIH transcription/DNA repair factor causes TTD-A
trichothiodystrophy disorder.` Nature Genet November
2000;26(3):307-13.
[0013] Fanconi Anaemia: Thyagarajan and Campbell `Elevated
homologous recombination activity in fanconi anemia fibroblasts.`:
J Biol Chem Sep. 12, 1997;272(37):23328-33.
[0014] Werner Syndrome: Kamath-Loeb et. al. `Functional interaction
between the Werner Syndrome protein and DNA polymerase delta.` Proc
Natl Acad Sci U S A Apr. 25, 2000;97(9):4603-8.
[0015] Breast cancer: Bertwistle et. al. `The pathology of familial
breast cancer How do the functions of BRCA1 and BRCA2 relate to
breast tumour pathology?` Breast Cancer Res 1999;1(1):41-47.
[0016] Lung cancer:Spitz et. al. `Modulation of nucleotide excision
repair capacity by XPD polymorphisms in lung cancer patients.`
Cancer Res Feb. 15, 2001;61(4):1354-7.
[0017] Skin cancer: Tomescu et. al. `Nucleotide excision repair
gene XPD polymorphisms and genetic predisposition to melanoma.`
Carcinogenesis March 2001;22(3):403-408.
[0018] Disruptions in DNA repair pathways have been found to be
involved in some of the most important genes concerned with cancer
such as p53, BRCA1 and BRCA2.
[0019] The complexities of the pathways leading to DNA repair allow
for many mechanisms by which it can be diverted. In addition to
genomic mutations, the epigenetic control of genes has been
implicated in disruptions to DNA repair pathways. The epigenetic
parameter that has been best characterised is DNA methylation.
Methylation of DNA repair genes has been implicated as a factor in
tumorigenesis `Promoter hypermethylation patterns of p16,
O6-methylguanine-DNA-methyltransferase, and death-associated
protein kinase in tumors and saliva of head and neck cancer
patients.` Cancer Res Feb. 1, 2001;61(3):939-42. The identification
of methylation of DNA repair genes as a factor in tumor malignancy
opens up the possibility of creating alternative methods of
treatment. Methylation based therapies could have considerable
advantages over current methods of treatment such as chemotherapy,
surgery and radiotherapy. Furthermore, as suggested by Rosas et.
al. DNA methylation analysis may provide novel means of tumor
diagnosis.
[0020] 5-methylcytosine is the most frequent covalent base
modification in the DNA of eukaryotic cells. It plays a role, for
example, in the regulation of the transcription, in genetic
imprinting, and in tumorigenesis. Therefore, the identification of
5-methylcytosine as a component of genetic information is of
considerable interest. However, 5-methylcytosine positions cannot
be identified by sequencing since 5-methylcytosine has the same
base pairing behavior as cytosine. Moreover, the epigenetic
information carried by 5-methylcytosine is completely lost during
PCR amplification.
[0021] A relatively new and currently the most frequently used
method for analyzing DNA for 5-methylcytosine is based upon the
specific reaction of bisulfite with cytosine which, upon subsequent
alkaline hydrolysis, is converted to uracil which corresponds to
thymidine in its base pairing behavior. However, 5-methylcytosine
remains unmodified under these conditions. Consequently, the
original DNA is converted in such a manner that methylcytosine,
which originally could not be distinguished from cytosine by its
hybridization behavior, can now be detected as the only remaining
cytosine using "normal" molecular biological techniques, for
example, by amplification and hybridization or sequencing. All of
these techniques are based on base pairing which can now be fully
exploited. In terms of sensitivity, the prior art is defined by a
method which encloses the DNA to be analyzed in an agarose matrix,
thus preventing the diffusion and renaturation of the DNA
(bisulfite only reacts with single-stranded DNA), and which
replaces all precipitation and purification steps with fast
dialysis (Olek A, Oswald J, Walter J. A modified and improved
method for bisulphite based cytosine methylation analysis. Nucleic
Acids Res. Dec. 15, 1996;24(24):5064-6). Using this method, it is
possible to analyze individual cells, which illustrates the
potential of the method. However, currently only individual regions
of a length of up to approximately 3000 base pairs are analyzed, a
global analysis of cells for thousands of possible methylation
events is not possible. However, this method cannot reliably
analyze very small fragments from small sample quantities either.
These are lost through the matrix in spite of the diffusion
protection.
[0022] An overview of the further known methods of detecting
5-methylcytosine may be gathered from the following review article:
Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998,
26, 2255.
[0023] To date, barring few exceptions (e.g., Zeschnigk M, Lich C,
Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the
diagnosis of Angelman and Prader-Willi syndrome based on allelic
methylation differences at the SNRPN locus. Eur J Hum Genet.
March-April 1997;5(2):94-8) the bisulfite technique is only used in
research. Always, however, short, specific fragments of a known
gene are amplified subsequent to a bisulfite treatment and either
completely sequenced (Olek A, Walter J. The pre-implantation
ontogeny of the H19 methylation imprint. Nat Genet. November
1997;17(3):275-6) or individual cytosine positions are detected by
a primer extension reaction (Gonzalgo M L, Jones P A. Rapid
quantitation of methylation differences at specific sites using
methylation-sensitive single nucleotide primer extension
(Ms-SNuPE). Nucleic Acids Res. Jun. 15, 1997;25(12):2529-31, WO
95/00669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a
sensitive and quantitative DNA methylation assay. Nucleic Acids
Res. Jun. 15, 1997;25(12):2532-4). In addition, detection by
hybridization has also been described (Olek et al., WO
99/28498).
[0024] Further publications dealing with the use of the bisulfite
technique for methylation detection in individual genes are: Grigg
G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA.
Bioessays. June 1994;16(6):431-6, 431; Zeschnigk M, Schmitz B,
Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments
in the human genome: different DNA methylation patterns in the
Prader-Willi/Angelman syndrome region as determined by the genomic
sequencing method. Hum Mol Genet. March 1997;6(3):387-95; Feil R,
Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on
individual chromosomes: improved protocol for bisulphite genomic
sequencing. Nucleic Acids Res. Feb. 25, 1994;22(4):695-6; Martin V,
Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing
indicates a correlation between DNA hypomethylation in the 5'
region of the pS2 gene and its expression in human breast cancer
cell lines. Gene. May 19, 1995;157(1-2):261-4; WO 97/46705, WO
95/15373 and WO 97/45560.
[0025] An overview of the Prior Art in oligomer array manufacturing
can be gathered from a special edition of Nature Genetics (Nature
Genetics Supplement, Volume 21, January 1999), published in January
1999, and from the literature cited therein.
[0026] Fluorescently labeled probes are often used for the scanning
of immobilized DNA arrays. The simple attachment of Cy3 and Cy5
dyes to the 5'-OH of the specific probe are particularly suitable
for fluorescence labels. The detection of the fluorescence of the
hybridized probes may be carried out, for example via a confocal
microscope. Cy3 and Cy5 dyes, besides many others, are commercially
available.
[0027] Matrix Assisted Laser Desorption Ionization Mass
Spectrometry (MALDI-TOF) is a very efficient development for the
analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption
ionization of proteins with molecular masses exceeding 10,000
daltons. Anal Chem. Oct. 15, 1988;60(20):2299-301). An analyte is
embedded in a light-absorbing matrix. The matrix is evaporated by a
short laser pulse thus transporting the analyte molecule into the
vapor phase in an unfragmented manner. The analyte is ionized by
collisions with matrix molecules. An applied voltage accelerates
the ions into a field-free flight tube. Due to their different
masses, the ions are accelerated at different rates. Smaller ions
reach the detector sooner than bigger ones.
[0028] MALDI-TOF spectrometry is excellently suited to the analysis
of peptides and proteins. The analysis of nucleic acids is somewhat
more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser
Desorption Ionization Mass Spectrometry. Current Innovations and
Future Trends. 1995, 1; 148-57). The sensitivity to nucleic acids
is approximately 100 times worse than to peptides and decreases
disproportionally with increasing fragment size. For nucleic acids
having a multiply negatively charged backbone, the ionization
process via the matrix is considerably less efficient. In MALDI-TOF
spectrometry, the selection of the matrix plays an eminently
important role. For the desorption of peptides, several very
efficient matrixes have been found which produce a very fine
crystallization. There are now several responsive matrixes for DNA,
however, the difference in sensitivity has not been reduced. The
difference in sensitivity can be reduced by chemically modifying
the DNA in such a manner that it becomes more similar to a peptide.
Phosphorothioate nucleic acids in which the usual phosphates of the
backbone are substituted with thiophosphates can be converted into
a chargeneutral DNA using simple alkylation chemistry (Gut IG, Beck
S. A procedure for selective DNA alkylation and detection by mass
spectrometry. Nucleic Acids Res. Apr. 25, 1995;23(8):1367-73). The
coupling of a charge tag to this modified DNA results in an
increase in sensitivity to the same level as that found for
peptides. A further advantage of charge tagging is the increased
stability of the analysis against impurities which make the
detection of unmodified substrates considerably more difficult.
[0029] Genomic DNA is obtained from DNA of cell, tissue or other
test samples using standard methods. This standard methodology is
found in references such as Fritsch and Maniatis eds., Molecular
Cloning: A Laboratory Manual, 1989.
DESCRIPTION
[0030] The object of the present invention is to provide the
chemically modified DNA of genes associated with DNA repair, as
well as oligonucleotides and/or PNA-oligomers for detecting
cytosine methylations, as well as a method which is particularly
suitable for the diagnosis and/or therapy of genetic and epigenetic
parameters of genes associated with DNA repair. The present
invention is based on the discovery that genetic and epigenetic
parameters and, in particular, the cytosine methylation pattern of
genes associated with DNA repair are particularly suitable for the
diagnosis and/or therapy of diseases associated with DNA
repair.
[0031] This objective is achieved according to the-present
invention using a nucleic acid containing a sequence of at least 18
bases in length of the chemically pretreated DNA of genes
associated with DNA repair according to one of Seq. ID No.1 through
Seq. ID No.144 and sequences complementary thereto and/or a
sequence of a chemically pretreated DNA of genes according to table
1 and sequences complementary thereto. In the table, after the
listed gene designations, the respective data bank numbers
(accession numbers) are specified which define the appertaining
gene sequences as unique. GenBank was used as the underlying data
bank, which is located at the National Institute of Health,
internet address www.ncbi.nlm.nih.gov.
[0032] The chemically modified nucleic acid could heretofore not be
connected with the ascertainment of genetic and epigenetic
parameters.
[0033] The object of the present invention is further achieved by
an oligonucleotide or oligomer for detecting the cytosine
methylation state in chemically pretreated DNA, containing at least
one base sequence having a length of at least 13 nucleotides which
hybridizes to a chemically pretreated DNA of genes associated with
DNA repair according to Seq. ID No.1 through Seq. ID No.144 and
sequences complementary thereto and/or a sequence of a chemically
pretreated DNA of genes according to table 1 and sequences
complementary thereto. The oligomer probes according to the present
invention constitute important and effective tools which, for the
first time, make it possible to ascertain the genetic and
epigenetic parameters of genes associated with DNA repair. The base
sequence of the oligomers preferably contain at least one CpG
dinucleotide. The probes may also exist in the form of a PNA
(peptide nucleic acid) which has particularly preferred pairing
properties. Particularly preferred are oligonucleotides according
to the present invention in which the cytosine of the CpG
dinucleotide is the 5.sup.th-9.sup.th nucleotide from the 5'-end of
the 13-mer; in the case of PNA-oligomers, it is preferred for the
cytosine of the CpG dinucleotide to be the 4.sup.th-6.sup.th
nucleotide from the 5'-end of the 9-mer.
[0034] The oligomers according to the present invention are
normally used in so called "sets" which contain at least one
oligomer for each of the CpG dinucleotides of the sequences of Seq.
ID No.1 through Seq. ID No.144 and sequences complementary thereto
and/or a sequence of a chemically pretreated DNA of genes according
to table 1 and sequences complementary thereto. Preferred is a set
which contains at least one oligomer for each of the CpG
dinucleotides from one of Seq. ID No.1 through Seq. ID No.144 and
sequences complementary thereto and/or a sequence of a chemically
pretreated DNA of genes according to table 1 and sequences
complementary thereto.
[0035] Moreover, the present invention makes available a set of at
least two oligonucleotides which can be used as so-called "primer
oligonucleotides" for amplifying DNA sequences of one of Seq. ID
No.1 through Seq. ID No.144 and sequences complementary thereto
and/or a sequence of a chemically pretreated DNA of genes according
to table 1 and sequences complementary thereto and fragments
thereof.
[0036] In the case of the sets of oligonucleotides according to the
present invention, it is preferred that at least one
oligonucleotide is bound to a solid phase.
[0037] The present invention moreover relates to a set of at least
10 n (oligonucleotides and/or PNA-oligomers) used for detecting the
cytosine methylation state in chemically pretreated genomic DNA
(Seq. ID No.1 through Seq. ID No.144 and sequences complementary
thereto and/or a sequence of a chemically pretreated DNA of genes
according to table 1 and sequences complementary thereto). These
probes enable diagnosis and/or therapy of genetic and epigenetic
parameters of genes associated with DNA repair. The set of
oligomers may also be used for detecting single nucleotide
polymorphisms (SNPs) in the chemically pretreated DNA of genes
associated with DNA repair according to one of Seq. ID No.1 through
Seq. ID No.144 and sequences complementary thereto and/or a
sequence of a chemically pretreated DNA of genes according to table
1 and sequences complementary thereto.
[0038] According to the present invention, it is preferred that an
arrangement of different oligonucleotides and/or PNA-oligomers (a
so-called "array") made available by the present invention is
present in a manner that it is likewise bound to a solid phase.
This array of different oligonucleotide- and/or PNA-oligomer
sequences can be characterized in that it is arranged on the solid
phase in the form of a rectangular or hexagonal lattice. The solid
phase surface is preferably composed of silicon, glass,
polystyrene, aluminium, steel, iron, copper, nickel, silver, or
gold. However, nitrocellulose as well as plastics such as nylon
which can exist in the form of pellets or also as resin matrices
are possible as well.
[0039] Therefore, a further subject matter of the present invention
is a method for manufacturing an array fixed to a carrier material
for analysis in connection with diseases associated with DNA repair
in which method at least one oligomer according to the present
invention is coupled to a solid phase. Methods for manufacturing
such arrays are known, for example, from U.S. Pat. No. 5,744,305 by
means of solid-phase chemistry and photolabile protecting
groups.
[0040] A further subject matter of the present invention relates to
a DNA chip for the analysis of diseases associated with DNA repair
which contains at least one nucleic acid according to the present
invention. DNA chips are known, for example, for U.S. Pat. No.
5,837,832.
[0041] Moreover, a subject matter of the present invention is a kit
which may be composed, for example, of a bisulfite-containing
reagent, a set of primer oligonucleotides containing at least two
oligonucleotides whose sequences in each case correspond or are
complementary to an 18 base long segment of the base sequences
specified in the appendix (Seq. ID No.1 through Seq. ID No.144 and
sequences complementary thereto and/or a sequence of a chemically
pretreated DNA of genes according to table 1 and sequences
complementary thereto), oligonucleotides and/or PNA-oligomers as
well as instructions for carrying out and evaluating the described
method. However, a kit along the lines of the present invention can
also contain only part of the aforementioned components.
[0042] The present invention also makes available a method for
ascertaining genetic and/or epigenetic parameters of genes
associated with the cycle cell by analyzing cytosine methylations
and single nucleotide polymorphisms, including the following
steps:
[0043] In the first step of the method, a genomic DNA sample is
chemically treated in such a manner that cytosine bases which are
unmethylated at the 5'-position are converted to uracil, thymine,
or another base which is dissimilar to cytosine in terms of
hybridization behavior. This will be understood as `chemical
pretreatment` hereinafter.
[0044] The genomic DNA to be analyzed is preferably obtained form
usual sources of DNA such as cells or cell components, for example,
cell lines, biopsies, blood, sputum, stool, urine, cerebral-spinal
fluid, tissue embedded in paraffin such as tissue from eyes,
intestine, kidney, brain, heart, prostate, lung, breast or liver,
histologic object slides, or combinations thereof.
[0045] The above described treatment of genomic DNA is preferably
carried out with bisulfite (hydrogen sulfite, disulfite) and
subsequent alkaline hydrolysis which results in a conversion of
non-methylated cytosine nucleobases to uracil or to another base
which is dissimilar to cytosine in terms of base pairing
behavior.
[0046] Fragments of the chemically pretreated DNA are amplified,
using sets of primer oligonucleotides according to the present
invention, and a, preferably heat-stable polymerase. Because of
statistical and practical considerations, preferably more than ten
different fragments having a length of 100-2000 base pairs are
amplified. The amplification of several DNA segments can be carried
out simultaneously in one and the same reaction vessel. Usually,
the amplification is carried out by means of a polymerase chain
reaction (PCR).
[0047] In a preferred embodiment of the method, the set of primer
oligonucleotides includes at least two olignonucleotides whose
sequences are each reverse complementary or identical to an at
least 18 base-pair long segment of the base sequences specified in
the appendix (Seq. ID No.1 through Seq. ID No.144 and sequences
complementary thereto and/or a sequence of a chemically pretreated
DNA of genes according to table 1 and sequences complementary
thereto). The primer oligonucleotides are preferably characterized
in that they do not contain any CpG dinucleotides.
[0048] According to the present invention, it is preferred that at
least one primer oligonucleotide is bonded to a solid phase during
amplification. The different oligonucleotide and/or PNA-oligomer
sequences can be arranged on a plane solid phase in the form of a
rectangular or hexagonal lattice, the solid phase surface
preferably being composed of silicon, glass, polystyrene,
aluminium, steel, iron, copper, nickel, silver, or gold, it being
possible for other materials such as nitrocellulose or plastics to
be used as well.
[0049] The fragments obtained by means of the amplification can
carry a directly or indirectly detectable label. Preferred are
labels in the form of fluorescence labels, radionuclides, or
detachable molecule fragments having a typical mass which can be
detected in a mass spectrometer, it being preferred that the
fragments that are produced have a single positive or negative net
charge for better detectability in the mass spectrometer. The
detection may be carried out and visualized by means of matrix
assisted laser desorption/ionization mass spectrometry (MALDI) or
using electron spray mass spectrometry (ESI).
[0050] The amplificates obtained in the second step of the method
are subsequently hybridized to an array or a set of
oligonucleotides and/or PNA probes. In this context, the
hybridization takes place in the manner described in the following.
The set of probes used during the hybridization is preferably
composed of at least 10 oligonucleotides or PNA-oligomers. In the
process, the amplificates serve as probes which hybridize to
oligonucleotides previously bonded to a solid phase. The
non-hybridized fragments are subsequently removed. Said
oligonucleotides contain at least one base sequence having a length
of 13 nucleotides which is reverse complementary or identical to a
segment of the base sequences specified in the appendix, the
segment containing at least one CpG dinucleotide. The cytosine of
the CpG dinucleotide is the 5.sup.th to 9.sup.th nucleotide from
the 5'-end of the 9-mer. One oligonucleotide exists for each CpG
dinucleotide. Said PNA-oligomers contain at least one base sequence
having a length of 9 nucleotides which is reverse complementary or
identical to a segment of the base sequences specified in the
appendix, the segment containing at least one CpG dinucleotide. The
cytosine of the CpG dinucleotide is the 4.sup.th to 6.sup.th
nucleotide seen from the 5'-end of the 9-mer. One oligonucleotide
exists for each CpG dinucleotide.
[0051] In the fourth step of the method, the non-hybridized
amplificates are removed. In the final step of the method, the
hybridized amplificates are detected. In this context, it is
preferred that labels attached to the amplificates are identifiable
at each position of the solid phase at which an oligonucleotide
sequence is located.
[0052] According to the present invention, it is preferred that the
labels of the amplificates are fluorescence labels, radionuclides,
or detachable molecule fragments having a typical mass which can be
detected in a mass spectrometer. The mass spectrometer is preferred
for the detection of the amplificates, fragments of the
amplificates or of probes which are complementary to the
amplificates, it being possible for the detection to be carried out
and visualized by means of matrix assisted laser
desorption/ionization mass spectrometry (MALDI) or using electron
spray mass spectrometry (ESI).
[0053] The produced fragments may have a single positive or
negative net charge for better detectability in the mass
spectrometer. The aforementioned method is preferably used for
ascertaining genetic and/or epigenetic parameters of genes
associated with DNA repair.
[0054] The oligomers according to the present invention or arrays
thereof as well as a kit according to the present invention are
intended to be used for the diagnosis and/or therapy of diseases
associated with DNA repair by analyzing methylation patterns of
genes associated with DNA repair. According to the present
invention, the method is preferably used for the diagnosis and/or
therapy of important genetic and/or epigenetic parameters within
genes associated with DNA repair.
[0055] The method according to the present invention is used, for
example, for the diagnosis and/or therapy of diseases.
[0056] The nucleic acids according to the present invention of Seq.
ID No.1 through Seq. ID No.144 and sequences complementary thereto
and/or a sequence of a chemically pretreated DNA of genes according
to table 1 and sequences complementary thereto can be used for the
diagnosis and/or therapy of genetic and/or epigenetic parameters of
genes associated with DNA repair.
[0057] The present invention moreover relates to a method for
manufacturing a diagnostic agent and/or therapeutic agent for the
diagnosis and/or therapy of diseases associated with DNA repair by
analyzing methylation patterns of genes associated with DNA repair,
the diagnostic agent and/or therapeutic agent being characterized
in that at least one nucleic acid according to the present
invention is used for manufacturing it, possibly together with
suitable additives and auxiliary agents.
[0058] A further subject matter of the present invention-relates to
a diagnostic agent and/or therapeutic agent for diseases associated
with DNA repair by analyzing methylation patterns of genes
associated with DNA repair, the diagnostic agent and/or therapeutic
agent containing at least one nucleic acid according to the present
invention, possibly together with suitable additives and auxiliary
agents.
[0059] The present invention moreover relates to the diagnosis
and/or prognosis of events which are disadvantageous to patients or
individuals in which important genetic and/or epigenetic parameters
within genes associated with DNA repair said parameters obtained by
means of the present invention may be compared to another set of
genetic and/or epigenetic parameters, the differences serving as
the basis for a diagnosis and/or prognosis of events which are
disadvantageous to patients or individuals.
[0060] In the context of the present invention the term
"hybridization" is to be understood as a bond of an oligonucleotide
to a completely complementary sequence along the lines of the
Watson-Crick base pairings in the sample DNA, forming a duplex
structure. To be understood by "stringent hybridization conditions"
are those conditions in which a hybridization is carried out at
60.degree. C. in 2.5.times.SSC buffer, followed by several washing
steps at 37.degree. C. in a low buffer concentration, and remains
stable.
[0061] The term "functional variants" denotes all DNA sequences
which are complementary to a DNA sequence, and which hybridize to
the reference sequence under stringent conditions and have an
activity similar to the corresponding polypeptide according to the
present invention.
[0062] In the context of the present invention, "genetic
parameters" are mutations and polymorphisms of genes associated
with DNA repair and sequences further required for their
regulation. To be designated as mutations are, in particular,
insertions, deletions, point mutations, inversions and
polymorphisms and, particularly preferred, SNPs (single nucleotide
polymorphisms).
[0063] In the context of the present invention, "epigenetic
parameters" are, in particular, cytosine methylations and further
chemical modifications of DNA bases of genes associated with DNA
repair and sequences further required for their regulation. Further
epigenetic parameters include, for example, the acetylation of
histones which, however, cannot be directly analyzed using the
described method but which, in turn, correlates with the DNA
methylation.
[0064] In the following, the present invention will be explained in
greater detail on the basis of the sequences and examples with
reference to the accompanying figure without being limited
thereto.
[0065] FIG. 1
[0066] FIG. 1 shows the hybridisation of fluorescent labelled
amplificates to a surface bound oligonucleotide. Sample I being
from healthy tissue and sample II being from olgodendrogliome
cerebrum (tumor) tissue. Fluorescence at a spot shows hybridisation
of the amplificate to the oligonucleotide. Hybridisation to a CG
oligonucleotide denotes methylation at the cytosine position being
analysed, hybridisation to a TG oligonucleotide denotes no
methylation at the cytosine position being analysed.
SEQ. ID NOS.1 to 144
[0067] Sequences having odd sequence numbers (e.g., Seq. ID No. 1,
3, 5, . . . ) exhibit in each case sequences of the chemically
pretreated genomic DNAs of different genes associated with DNA
repair.Sequences having even sequence numbers (e.g., Seq. ID No. 2,
4, 6, . . . ) exhibit in each case the sequences of the chemically
pretreated genomic DNAs of genes associated with DNA repair which
are complementary to the preceeding sequences (e.g., the
complementary sequence to Seq. ID No.1 is Seq. ID No.2, the
complementary sequence to Seq. ID No.3 is Seq. ID No.4, etc.)
Seq. ID Nos. 145 to 148
[0068] Seq. ID Nos. 145 to 148 show the sequences of
oligonucleotides used in Example 1.
[0069] The following example relates to a fragment of a gene
associated with DNA repair, in this case, Uracil-DNA glycosylase
(UNG) in which a specific CG-position is analyzed for its
methylation status.
EXAMPLE 1
Methylation Analysis in the Gene Uracil-DNA Glycosylase (UNG)
Associated with DNA Repair
[0070] The following example relates to a fragment of the gene UNG
in which a specific CG-position is to be analyzed for
methylation.
[0071] In the first step, a genomic sequence is treated using
bisulfite (hydrogen sulfite, disulfite) in such a manner that all
cytosines which are not methylated at the 5-position of the base
are modified in such a manner that a different base is substituted
with regard to the base pairing behavior while the cytosines
methylated at the 5-position remain unchanged.
[0072] If bisulfite solution is used for the reaction, then an
addition takes place at the non-methylated cytosine bases.
Moreover, a denaturating reagent or solvent as well as a radical
interceptor must be present. A subsequent alkaline hydrolysis then
gives rise to the conversion of non-methylated cytosine nucleobases
to uracil. The chemically converted DNA (sequence ID 73) is then
used for the detection of methylated cytosines. In the second
method step, the treated DNA sample is diluted with water or an
aqueous solution. Preferably, the DNA is subsequently desulfonated
(10-30 min, 90-100.degree. C.) at an alkaline pH value. In the
third step of the method, the DNA sample is amplified in a
polymerase chain reaction, preferably using a heat-resistant DNA
polymerase. In the present case, cytosines of the gene UNG are
analyzed. To this end, a defined fragment having a length of 476 bp
is amplified with the specific primer oligonucleotides
GTTATAGTTATAGTTAGGGT (Sequence ID No. 145) and TCTCCCCTCTAATTAAACAA
(Sequence ID No. 146). This amplificate serves as a sample which
hybridizes to an oligonucleotide previously bonded to a solid
phase, forming a duplex structure, for example AGGAAGGCGGTGGGTTT
(Sequence ID No. 147), the cytosine to be detected being located at
position 252 of the amplificate. The detection of the hybridization
product is based on Cy3 and Cy5 fluorescently labeled primer
oligonucleotides which have been used for the amplification. A
hybridization reaction of the amplified DNA with the
oligonucleotide takes place only if a methylated cytosine was
present at this location in the bisulfite-treated DNA. Thus, the
methylation status of the specific cytosine to be analyzed is
inferred from the hybridization product.
[0073] In order to verify the methylation status of the position, a
sample of the amplificate is further hybridized to another
oligonucleotide previously bonded to a solid phase. Said
olignonucleotide is identical to the oligonucleotide previously
used to analyze the methylation status of the sample, with the
exception of the position in question. At the position to be
analysed said oligonucleotide comprises a thymine base as opposed
to a cytosine base i.e AGGAAGGTGGTGGGTTT (Sequence ID No. 148).
Therefore, the hybridisation reaction only takes place if an
unmethylated cytosine was present at the position to be analysed.
The procedure was carried out on cell samples from 2 patients,
sample I being from normal healthy tissue and sample II being from
a olgodendrogliome cerebrum tumor sample.
[0074] From the results (FIG. 1) it can be seen that Sample I was
methylated and sample II was unmethylated.
EXAMPLE 2
Diagnosis of Diseases Associated with DNA Repair
[0075] In order to relate the methylation patterns to one of the
diseases associated with DNA repair, it is initially required to
analyze the DNA methylation patterns of a group of diseased and of
a group of healthy patients. These analyses are carried out, for
example, analogously to Example 1. The results obtained in this
manner are stored in a database and the CpG dinucleotides which are
methylated differently between the two groups are identified. This
can be carried out by determining individual CpG methylation rates
as can be done, for example, in a relatively imprecise manner, by
sequencing or else, in a very precise manner, by a
methylation-sensitive "primer extension reaction". It is also
possible for the entire methylation status to be analyzed
simultaneously, and for the patterns to be compared, for example,
by clustering analyses which can be carried out, for example, by a
computer.
[0076] Subsequently, it is possible to allocate the examined
patients to a specific therapy group and to treat these patients
selectively with an individualized therapy.
[0077] Example 2 can be carried, out, for example, for the
following diseases: Ataxia telangiectasia, Ageing, Bloom's
Syndrome, Immunodeficiency, Cockayne syndrome, Nijmegen breakage
syndrome, Trichothiodystrophy, Fanconi Anaemia, Werner Syndrome,
solid tumors and cancer
1TABLE I Listing of particularly preferred genes of the present
invention associated with DNA repair Database Entry (Genbank,
internet Gene address www.ncbi.nlm.nih.gov) PMS2L1 D38435 PMS2L12
AF053356 PMS2L2 D38436 PMS2L3 D38437 PMS2L4 D38438 and D38500
PMS2L5 D38439 PMS2L6 D38440 MGMT NM_002412 MSH2 NM_000251 NUDT1
NM_002452 TDG NM_003211 INPPL1 NM_001567 RFC4 NM_002916 DDIT1L
FANCB XRCC8
[0078]
Sequence CWU 0
0
* * * * *
References