U.S. patent application number 10/240453 was filed with the patent office on 2003-08-07 for diagnosis of diseases associated with dna transcription.
Invention is credited to Berlin, Kurt, Olek, Alexander, Piepenbrock, Christian.
Application Number | 20030148326 10/240453 |
Document ID | / |
Family ID | 27437807 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148326 |
Kind Code |
A1 |
Olek, Alexander ; et
al. |
August 7, 2003 |
Diagnosis of diseases associated with dna transcription
Abstract
The present invention relates to the chemically modified genomic
sequences of genes associated with DNA transcription to
oligonucleotides and/or PNA-oligomers for detecting the cytosine
methylation state of genes associated with DNA transcription which
are directed against the sequence as well as to a method for
ascertaining genetic and/or epigenetic parameters of genes
associated with DNA transcription.
Inventors: |
Olek, Alexander; (Berlin,
DE) ; Piepenbrock, Christian; (Berlin, DE) ;
Berlin, Kurt; (Stahnsdorf, DE) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Family ID: |
27437807 |
Appl. No.: |
10/240453 |
Filed: |
January 21, 2003 |
PCT Filed: |
April 6, 2001 |
PCT NO: |
PCT/EP01/03973 |
Current U.S.
Class: |
435/6.12 ;
435/287.2; 530/350; 536/24.3 |
Current CPC
Class: |
C07K 14/4703 20130101;
A61P 7/04 20180101; C12Q 2600/154 20130101; C12Q 1/6883 20130101;
C12Q 2600/156 20130101; A61P 13/12 20180101; A61P 35/00 20180101;
A61P 11/06 20180101; C07K 14/82 20130101; A61P 29/00 20180101; C12Q
1/6886 20130101; A61P 9/10 20180101 |
Class at
Publication: |
435/6 ;
435/287.2; 530/350; 536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04; C12M 001/34; C07K 014/00 |
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 transcription according to one of the sequences taken from
the group of Seq. ID No.1 to Seq. ID No.346 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 transcription according to a sequence according
to one of the genes ELF1 (M82882), ETV3 (L16464), ETV4 (D12765),
TAF2C2 (Y09321), TCF9 (M29204), ZNF121 (M99593), ZNF131 (U09410),
ZNF139 (U09848), ZNF154 (U20648), ZNF169 (U28322), ZNF2 (X60152),
ZNF204 (AF033199), ZNF206 (AA206569), ZNF3 (X78926), ZNF37A
(X69115), ZNF44 (X16281), ZNF8 (M29581), ZK1 (NM.sub.--005815), ADA
(NM.sub.--000022), ATBF1 ((NM.sub.--006885), ATF3
(NM.sub.--001674), ZNF255 (NM.sub.--005774), CBFB(NM.sub.--001755),
CEBPD (NM.sub.--005195), CTPS (NM.sub.--001905), DCTD
(NM.sub.--001921), DR1 (NM.sub.--001938), ELF3 (NM.sub.--004433),
ELK4 (NM.sub.--021795), ETV5 (NM.sub.--004454), KLF4
(NM.sub.--004235), FOXO1A (NM.sub.--002015), FLI1
(NM.sub.--002017), HHEX (NM.sub.--001529), HIVEP1
(NM.sub.--002114), HMG2 (NM.sub.--002129), ID1 (NM.sub.--002165),
ID3 (NM.sub.--002167), LAF4 (NM.sub.--002285), ZNFN1A1
(NM.sub.--006060), LYL1 (NM.sub.--005583), MAFG (NM.sub.--002359),
MAZ (NM.sub.--002383), ODC1 (NM.sub.--002539), PBX3
(NM.sub.--006195), POU2AF1 (NM.sub.--006235), POU2F2
(NM.sub.--002698), POU3F1 (NM.sub.--002699), PROP1
(NM.sub.--006261), RARG (NM.sub.--000966), RECQL (NM.sub.--002907),
RXRA (NM.sub.--002957), SP100 (NM.sub.--003113), ZFP93
(NM.sub.--004234), MKRN3 (NM.sub.--005664), ZNF133
(NM.sub.--003434), ZNF157 (NM.sub.--003446), ZNF173
(NM.sub.--003449), ZNF189 (NM.sub.--003452), ZNF207
(NM.sub.--003457), ZNF262 (NM.sub.--005095), ZNF264
(NM.sub.--003417), ZNF74 (NM.sub.--003426), ZNF91
(NM.sub.--003430), POU3F2 (NM.sub.--005604), ZNF84
(NM.sub.--003428), ERV3, TCF8 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 transcription according to one of the Seq
ID Nos 1 to 346 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 346
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. 346
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. 346
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
Adenosine deaminase deficiency, Viral infection, Retroviral
infection, Sezary syndrome, Hematological disorders, Immunological
disorders, Werner syndrome, Tuberculosis, Developmental disorders,
Psoriasis, Rieger syndrome, Neurological disorders,
Neurodegenerative disorders, Waardenburg syndrome, Niemann-Pick
disease, Myelodysplastic syndrome, Myocardial infarction,
Hypertension, Angiogenesis, Erythropoiesis, Congenital heart
disease, HDR syndrome, Myelodysplastic syndrome, Arthiritis,
Polyglutamine disorders, 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 Adenosine
deaminase deficiency, Viral infection, Retroviral infection, Sezary
syndrome, Hematological disorders, Immunological disorders, Werner
syndrome, Tuberculosis, Developmental disorders, Psoriasis, Rieger
syndrome, Neurological disorders, Neurodegenerative disorders,
Waardenburg syndrome, Niemann-Pick disease, Myelodysplastic
syndrome, Myocardial infarction, Hypertension, Angiogenesis,
Erythropoiesis, Congenital heart disease, HDR syndrome,
Myelodysplastic syndrome, Arthiritis, Polyglutamine disorders,
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 in of the development of an
individual, and how the activation and inhibition of specific genes
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
transcription and, in particular, with the methylation status
thereof.
PRIOR ART
[0003] DNA transcription, the process of using genomic DNA as a
template for the synthesis of RNA is a complex process. It requires
the interaction of various factors, enzymes and sequences. The
initiation of DNA transcription requires a complex known as the
basal transcription apparatus consisting of RNA polymerase combined
with various factors. They form a complex at the startpoint from
which transcription proceeds. Examples of such factors include
TFIID, TRF1 and TRF2. This is a major control point for gene
expression.
[0004] Transcription factors are often classified according to
their DNA binding domain. Members of the same group have sequence
variations of a specific motif that confer specificity for
individual target sites. Common categories include, steroid
receptors, zinc finger motifs, helix-turn-helix motifs,
helix-loop-helix motifs and leucine zippers.
[0005] The transcription activity is further supplemented by the
binding of other components, such as upstream and inducible
factors. DNA sequence components of the system include both
promoter and enhancer elements. Promoter sequences are located in
the vicinity of the transcribed region and enhancer elements are
located at a distance from the startpoint. These sequences interact
with the factors mentioned above to regulate the transcription of
RNA.
[0006] Further control may be exerted by the action of insulator
elements, specialised chromatin structures that have hypersensitive
sites. They are able to block passage of any activating or
inactivating effects from enhancers, silencers, or LCRs.
[0007] A further requirement of DNA transcription is that the DNA
be accessible. In most cases the initiation of transcription
requires that the DNA be free of nucleosomes. Therefore, a
transcription factor, or some other nonhistone protein concerned
with the particular function of the site, modifies the properties
of a short region of DNA so that nucleosomes are excluded. Several
chromatin modification mechanisms are utilised, including histone
acetylation and deacetylation activity.
[0008] A further parameter that regulates genomic transcription is
methylation. Methylation of CpG islands in the regulatory regions
of genes has been shown to be a common method of transcription
regulation. Furthermore, aberrant methylation patterns have been
associated with a variety of disease phenotypes. For example:
[0009] Head and neck cancer: Sanchez-Cespedes M et al. `Gene
promoter hypermethylation in tumours and serum of head and neck
cancer patients` Cancer Res. Feb. 15, 2000;60 (4):892-5;
[0010] Hodgkin's disease: Garcia J F et al `Loss of p16 protein
expression associated with methylation of the p16INK4A gene is a
frequent finding in Hodgkin's disease` Lab invest December 1999;79
(12):1453-9;
[0011] Gastric cancer: Yanagisawa Y et al. `Methylation of the
hMLH1 promoter in familial gastric cancer with microsatellite
instability` Int J Cancer Jan. 1, 2000; 85 (1):50-3;
[0012] Prader-Willi/Angelman's syndrome: Zeschnigh et al `Imprinted
segments in the human genome: different DNA methylation patterns in
the Prader Willi/Angelman syndrome region as determined by the
genomic sequencing method` Human Mol. Genetics (1997) (6) 3 pp
387-395;
[0013] ICF syndrome: Tuck-Muller et al `CMDNA hypomethylation and
unusual chromosome instability in cell lines from ICF syndrome
patients` Cytogenet Call Genet 2000; 89(1-2):121-8;
[0014] Dermatofibroma: Chen T C et al `Dermatofibroma is a clonal
proliferative disease` J Cutan Pathol January 2000;27
(1):36-9);
[0015] Hypertension: Lee S D et al. `Monoclonal endothelial cell
proliferation is present in primary but not secondary pulmonary
hypertension` J clin Invest Mar. 1, 1998, 101 (5):927-34;
[0016] Autism: Klauck S M et al. `Molecular genetic analysis of the
FMR-1 gene in a large collection of autistic patients` Human Genet
August 1997; 100 (2): 224-9;
[0017] Fragile X Syndrome: Hornstra I K et al. `High resolution
methylation analysis of the FMR1 gene trinucleotide repeat region
in fragile X syndrome` Hum Mol Genet October 1993,
2(10):1659-65;
[0018] Huntigton's disease: Ferluga J et al. `possible organ and
age related epigenetic factors in Huntington's disease and
colorectal carcinoma` Med hypotheses May 1989;29(1);51-4;
[0019] The proper functioning of the DNA transcription system is
essential for the maintenance of cellular functions. Disruptions to
the ordered transcription of DNA may impact on a wide variety of
disease phenotypes. Mutations in sequences coding for and
comprising essential components of the DNA transcription system
e.g. zinc finger proteins and enhancer elements have been
implicated in a variety of disorders, including cancer:
[0020] Adenosine deaminase deficiency: Ariga T `Gene therapy for
adenosine deaminase (ADA) deficiency: review of the past, the
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[0021] Cancer: Garte S, Sogawa K `Ah receptor gene polymorphisms
and human cancer susceptibility` IARC Sci Publ
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[0022] Hodgkin's disease: Sandvej K, Andresen B S, Zhou X G,
Gregersen N, Hamilton-Dutoit S `Analysis of the Epstein-Barr virus
(EBV) latent membrane protein 1 (LMP-1) gene and promoter in
Hodgkin's disease isolates: selection against EBV variants with
mutations in the LMP-1 promoter ATF-1/CREB-1 binding site` Mol
Pathol October 2000;53(5):280-8;
[0023] Ewing's sarcoma family tumors: Aryee D N, Sommergruber W,
Muehlbacher K, Dockhorn-Dworniczak B, Zoubek A, Kovar H
`Variability in gene expression patterns of Ewing tumor cell lines
differing in EWS-FLI1 fusion type` Lab Invest December
2000;80(12):1833-44;
[0024] Colon cancer: Ishiguro T, Nagawa H, Naito M, Tsuruo T Jpn J
`Inhibitory effect of ATF3 antisense oligonucleotide on ectopic
growth of HT29 human colon cancer cells` Cancer Res August
2000;91(8):833-6;
[0025] Breast cancer: Irminger-Finger I, Siegel B D, Leung W C `The
functions of breast cancer susceptibility gene 1 (BRCA1) product
and its associated proteins` Biol Chem February 1999;
380(2):117-28;
[0026] Endometrial cancer: Hori M, Takechi K, Arai Y, Yomo H,
Itabashi M, Shimazaki J, Inagawa S, Hori M `Comparison of
macroscopic appearance and estrogen receptor-alpha regulators after
gene alteration in human endometrial cancer` Int J Gynecol Cancer
November 2000; 10(6):469-476;
[0027] Wilms' tumor: Inoue K, Sugiyama H, Ogawa H, Nakagawa M,
Yamagami T, Miwa H, Kita K, Hiraoka A, Masaoka T, Nasu K, et al
`WT1 as a new prognostic factor and a new marker for the detection
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[0028] Solid tumors: Kok K, Naylor S L, Buys C H `Deletions of the
short arm of chromosome 3 in solid tumors and the search for
suppressor genes` Adv Cancer Res 1997;71:27-92;
[0029] Leukemia; Tanaka T, Kurokawa M, Ueki K, Tanaka K, Imai Y,
Mitani K, Okazaki K, Sagata N, Yazaki Y, Shibata Y, Kadowaki T,
Hirai H `The extracellular signal-regulated kinase pathway
phosphorylates AML1, an acute myeloid leukemia gene product, and
potentially regulates its transactivation ability`Mol Cell Biol
July 1996;16(7):3967-79;
[0030] Polyglutamine disorders: Nucifora F C Jr, Sasaki M, Peters M
F, Huang H, Cooper J K, Yamada M, Takahashi H, Tsuji S, Troncoso J,
Dawson V L, Dawson T M, Ross C A `Interference by huntingtin and
atrophin-1 with cbp-mediated transcription leading to cellular
toxicity` Science Mar. 23, 2001;291(5512):2423-8;
[0031] Rheumatoid arthritis: Pascual M, Nieto A, Lopez-Nevot M A,
Ramal L, Mataran L, Caballero A, Alonso A, Martin J, Zanelli E
`Rheumatoid arthritis in southern Spain: toward elucidation of a
unifying role of the HLA class II region in disease predisposition`
Arthritis Rheum February 2001;44(2):307-14;
[0032] Neurodegenerative disorders: Mattila K M, Axelman K, Rinne J
O, Blomberg M, Lehtimaki T, Laippala P, Roytta M, Viitanen M,
Wahlund L, Winblad B, Lannfelt L `Interaction between estrogen
receptor 1 and the epsilon4 allele of apolipoprotein E increases
the risk of familial Alzheimer's disease in women` Neurosci Lett
Mar. 17, 2000;282(1-2):45-8;
[0033] Myelodysplastic syndrome and acute leukemia: Patmasiriwat P,
Fraizer G, Kantarjian H, Saunders G F `WT1 and GATA1 expression in
myelodysplastic syndrome and acute leukemia` Leukemia June
1999;13(6):891-900;
[0034] HDR syndrome: Van Esch H, Groenen P, Nesbit M A,
Schuffenhauer S, Lichtner P, Vanderlinden G, Harding B, Beetz R,
Bilous R W, Holdaway I, Shaw N J, Fryns J P, Van de Ven W, Thakker
R V, Devriendt K `GATA3 haplo-insufficiency causes human HDR
syndrome` Nature Jul. 27, 2000;406(6794):419-22;
[0035] Congenital heart disease: Pehlivan T, Pober B R, Brueckner
M, Garrett S, Slaugh R, Van Rheeden R, Wilson D B, Watson M S, Hing
A V`GATA4 haploinsufficiency in patients with interstitial deletion
of chromosome region 8p23.1 and congenital heart disease` Am J Med
Genet Mar. 19, 1999;83(3):201-6;
[0036] Angiogenesis and erythropoiesis: Krieg M, Haas R, Brauch H,
Acker T, Flamme I, Plate K H `Up-regulation of hypoxia-inducible
factors HIF-1alpha and HIF-2alpha under normoxic conditions in
renal carcinoma cells by von Hippel-Lindau tumor suppressor gene
loss of function Oncogene Nov. 16, 2000;19(48):5435-43;
[0037] Hypertension: Morse J H, Barst R J, Fotino M, Zhang Y,
Flaster E, Fritzler M J `Primary pulmonary hypertension:
immunogenetic response to high-mobility group(HMG) proteins and
histone` Clin Exp Immunol November 1996;106(2):389-95;
[0038] Myocardial infarction: Hegele R A, Huang L S, Herbert P N,
Blum C B, Buring J E, Hennekens C H, Breslow J L `Apolipoprotein
B-gene DNA polymorphisms associated with myocardial infarction.` N
Engl J Med Dec. 11, 1986;315(24):1509-15;
[0039] Early onset pauciarticular chronic arthritis: Epplen C,
Rumpf H, Albert E, Haas P, Truckenbrodt H, Epplen J T
`Immunoprinting excludes many potential susceptibility genes as
predisposing to early onset pauciarticular juvenile chronic
arthritis except HLA class II and TNF` Eur J Immunogenet August
1995;22(4):311-22;
[0040] AIDS-related non-Hodgkin lymphomas and Hodgkin lymphomas
arising in HIV+ patients: Carbone A, Gloghini A, Larocca L M,
Capello D, Pierconti F, Canzonieri V, Tirelli U, Dalla-Favera
R,Gaidano G `Expression profile of MUM1/IRF4, BCL-6, and
CD138/syndecan-1 defines novel histogenetic subsets of human
immunodeficiency virus-related lymphomas` Blood Feb. 1,
2001;97(3):744-51;
[0041] Multiple endocrine neoplasia: Calender A `Molecular genetics
of neuroendocrine tumors` Digestion 2000;62 Suppl 1:3-18;
[0042] Myelodysplastic syndrome and acute myeloid leukemia: Xie J,
Briggs J A, Morris S W, Olson M O, Kinney M C, Briggs R C `MNDA
binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5)
associated with myelodysplastic syndrome and acute myeloid
leukemia` Exp Hematol October 1997;25(11):111-7;
[0043] Niemann-Pick type C2 disease: Naureckiene S, Sleat D E,
Lackland H, Fensom A, Vanier M T, Wattiaux R, Jadot M, Lobel P
`Identification of HE1 as the second gene of Niemann-Pick C
disease` Science Dec. 22, 2000;290(5500):2298-301;
[0044] Waardenburg syndrome: Bondurand N, Pingault V, Goerich D E,
Lemort N, Sock E, Caignec C L, Wegner M, Goossens M `Interaction
among SOX10, PAX3 and MITF, three genes altered in Waardenburg
syndrome` Hum Mol Genet Aug. 12, 2000;9(13):1907-17;
[0045] Neurological disorders: Geisbrecht B V, Collins C S, Reuber
B E, Gould S J `Disruption of a PEX1-PEX6 interaction is the most
common cause of the neurologic disorders Zellweger syndrome,
neonatal adrenoleukodystrophy, and infantile Refsum disease` Proc
Natl Acad Sci USA Jul. 21, 1998;95(15):8630-5;
[0046] Rieger syndrome: Hjalt T, Amendt B, Murray J `PITX2
Regulates Procollagen Lysyl Hydroxylase (PLOD) Gene Expression.
Implications for the pathology of rieger syndrome` J Cell Biol Feb.
5, 2001;152(3):545-52;
[0047] Psoriasis vulgaris: Oka A, Tamiya G, Tomizawa M, Ota M,
Katsuyama Y, Makino S, Shiina T, Yoshitome M, Ezuka M, Sasao Y,
Iwashita K, Kawakubo Y, Sugai J, Ozawa A, Ohkido M, Kimura M,
Bahram S, Inoko H `Association analysis using refined
microsatellite markers localizes a susceptibility locus for
psoriasis vulgaris within a 111 kb segment telomeric to the HLA-C
gene` Hum Mol Genet November 1999;8(12):2165-70;
[0048] Developmental disorders: Dattani M T, Robinson I C `The
molecular basis for developmental disorders of the pituitary gland
in man` Clin Genet May 2000;57(5):337-46;
[0049] Tuberculosis: Selvaraj P, Narayanan P R, Reetha A M
`Association of vitamin D receptor genotypes with the
susceptibility to pulmonary tuberculosis in female patients &
resistance in female contacts` Indian J Med Res May
2000;111:172-9;
[0050] Werner syndrome: Kitao S, Lindor N M, Shiratori M, Furuichi
Y, Shimamoto A `Rothmund-thomson syndrome responsible gene, RECQL4:
genomic structure and products` Genomics Nov. 1,
1999;61(3):268-76;
[0051] Immunological disorders: Puglatti et. al. `The genes for MHC
class II regulatory factors RFX1 and RFX2 are located on the short
arm of chromosome 19` Genomics August 1992; 13(4):1307-10;
[0052] Hematological disorders: Elbein S C, Teng K, Eddings K,
Hargrove D, Scroggin E `Molecular scanning analysis of hepatocyte
nuclear factor 1alpha (TCF1) gene in typical familial type 2
diabetes in African Americans` Metabolism February
2000;49(2):280-4;
[0053] Sezary syndrome: Showe L C, Fox F E, Williams D, Au K, Niu
Z, Rook A H `Depressed IL-12-mediated signal transduction in T
cells from patients with Sezary syndrome is associated with the
absence of IL-12 receptor beta 2 mRNA and highly reduced levels of
STAT4` J Immunol Oct. 1, 1999;163(7):4073-9;
[0054] Viral infection: Yoshida M `HTLV-1 oncoprotein Tax
deregulates transcription of cellular genes through multiple
mechanisms` J Cancer Res Clin Oncol 1995;121(9-10):521-8;
[0055] The large number of components involved in DNA transcription
provide novel targets for therapies and diagnosis for diseases. In
particular this may be relevant to diseases where current therapies
may have unwanted side effects or fail to provide effective
treatment. For cancer patients such methods constitute a
considerable advantage over conventional methods such as
chemotherapy, which with their massive side effects, sometimes
result in unacceptable morbidity or lead up to the death of the
patient. In practice, the unwanted side effects associated with
cancer therapies frequently limit the treatment which could help a
patient.
[0056] A global analysis of the status of DNA transcription
mechanisms would provide a basis for the development of appropriate
and specific therapies for diseases associated with DNA
replication. The current state of the art is such that the analysis
may be carried out in a gene specific manner based on the results
of gene expression, e.g. DNA micro array analysis of mRNA
expression or proteomic analysis. The next step would then be to
look at the causal factors involved at earlier stages in the
regulatory mechanisms controlling DNA transcription. DNA
methylation provides a novel level of information at which to
analyse the genome.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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):252.9-31, 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).
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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, 1998;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.
[0065] 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; 350-57). 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 charge-neutral DNA using simple alkylation chemistry (Gut I G,
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.
[0066] 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.
[0067] Description
[0068] The object of the present invention is to provide the
chemically modified DNA of genes associated with DNA transcription,
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 transcription. 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 transcription are particularly suitable
for the diagnosis and/or therapy of diseases associated with DNA
transcription.
[0069] 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 transcription according to one of Seq. ID No.1
through Seq. ID No.346 and sequences complementary thereto and/or a
chemically pretreated DNA of genes according to the sequences 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 at internet address http://www.ncbi.nlm.nih.gov.
[0070] The chemically modified nucleic acid could heretofore not be
connected with the ascertainment of genetic and epigenetic
parameters.
[0071] 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 transcription according to Seq. ID No.1 through Seq. ID No.346
and sequences complementary thereto and/or a chemically pretreated
DNA of genes according to the sequences 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 transcription. 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 referred
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.
[0072] 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.346 and sequences complementary thereto
and/or a chemically pretreated DNA of genes according to the
sequences 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.346 and sequences complementary thereto and/or a
chemically pretreated DNA of genes according to the sequences of
genes according to table 1 and sequences complementary thereto.
[0073] 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.346 and sequences complementary thereto
and/or a chemically pretreated DNA of genes according to the
sequences of genes according to table 1 and sequences complementary
thereto, or segments thereof.
[0074] 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.
[0075] 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.346 and sequences complementary
thereto and/or a chemically pretreated DNA of genes according to
the sequences 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 transcription. The set of oligomers may also be used for
detecting single nucleotide polymorphisms (SNPs) in the chemically
pretreated DNA of genes associated with DNA transcription according
to one of Seq. ID No.1 through Seq. ID No.346 and sequences
complementary thereto and/or a chemically pretreated DNA of genes
according to the sequences of genes according to table 1 and
sequences complementary thereto.
[0076] 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.
[0077] 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
transcription 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.
[0078] A further subject matter of the present invention relates to
a DNA chip for the analysis of diseases associated with DNA
transcription 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.
[0079] 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.346 and
sequences complementary thereto and/or a chemically pretreated DNA
of genes according to the sequences 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.
[0080] 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:
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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).
[0085] 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.346 and sequences
complementary thereto and/or a chemically pretreated DNA of genes
according to the sequences 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.
[0086] 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.
[0087] 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).
[0088] 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 13-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.
[0089] In the fourth step of the method, the non-hybridized
amplificates are removed.
[0090] 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.
[0091] 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).
[0092] 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 transcription.
[0093] 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 transcription by analyzing methylation patterns
of genes associated with DNA transcription. 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 transcription.
[0094] The method according to the present invention is used, for
example, for the diagnosis and/or therapy of diseases.
[0095] The nucleic acids according to the present invention of Seq.
ID No.1 through Seq. ID No.346 and sequences complementary thereto
and/or a chemically pretreated DNA of genes according to the
sequences 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
transcription.
[0096] 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
transcription by analyzing methylation patterns of genes associated
with DNA transcription, 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.
[0097] A further subject matter of the present invention relates to
a diagnostic agent and/or therapeutic agent for diseases associated
with DNA transcription by analyzing methylation patterns of genes
associated with DNA transcription, 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.
[0098] 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 transcription 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.
[0099] 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.
[0100] 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.
[0101] In the context of the present invention, "genetic
parameters" are mutations and polymorphisms of genes associated
with DNA transcription 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).
[0102] 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
transcription 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.
[0103] In the following, the present invention will be explained in
greater detail on the basis of the sequences and examples with
respect to the accompanying FIGURE without being limited
thereto.
[0104] FIG. 1
[0105] FIG. 1 shows the hybridisation of fluorescent labelled
amplificates to a surface bound olignonucleotide. Sample I being
from a pilocytic astrocytoma tumor sample and sample II being form
an oligodenrogliome grade II tumor sample. Flourescence at a spot
shows hybridisation of the amplificate to the olignonucleotide.
Hybridisation to a CG olgnonucleotide denotes methylation at the
cytosine position being analysed, gybridisation to a TG
olignonucleotide denates no methylation at the cytosine position
being analysed.
[0106] Seq. ID No. 1 Through Seq. ID No. 346
[0107] 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
transcription. 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
transcription 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.)
[0108] Seq. ID No. 347 Through Seq. ID No. 350
[0109] Seq. ID No. 347 through Seq. ID No. 350 show sequences of
oligonucleotides used in Example 1.
[0110] The following example relates to a fragment of a gene
associated with DNA transcription, in this case, CFOS in which a
specific CG-position is analyzed for its methylation status.
EXAMPLE 1
Methylation Analysis in the Gene CFOS Associated with DNA
Transcription
[0111] The following example relates to a fragment of the gene CFOS
in which a specific CG-position is to be analyzed for
methylation.
[0112] 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.
[0113] 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 345) 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 CFOS are
analyzed. To this end, a defined fragment having a length of 951 bp
is amplified with the specific primer oligonucleotides
TTTTGAGTTTTAGAATTGTT (Sequence ID No. 347) and AAAAACCCCCTACTCATCTA
(Sequence ID No. 348). This amplificate serves as a sample which
hybridizes to an oligonucleotide previously bonded to a solid
phase, forming a duplex structure, for example AAAACATTCGCACCTAAT
(Sequence ID No. 349), the cytosine to be detected being located at
position 105. 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.
[0114] 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
oligonucleotide 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 AAAACATTCACACCTAAT (Sequence ID No. 350). 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 a
pilocytic astrocytoma tumor sample and sample II being from an
oligodenrogliome grade II tumor sample.
[0115] From the results (FIG. 1) it can be seen that Sample I
contained both methylated and unmethylated cells and sample II was
unmethylated.
EXAMPLE 2
Diagonosis of Diseases Associated with DNA Transcription
[0116] In order to relate the methylation patterns to one of the
diseases associated with DNA transcription, 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.
[0117] Subsequently, it is possible to allocate the examined
patients to a specific therapy group and to treat these patients
selectively with an individualized therapy.
[0118] Example 2 can be carried out, for example, for the following
diseases: Adenosine deaminase deficiency, Viral infection,
Retroviral infection, Sezary syndrome, Hematological disorders,
Immunological disorders, Werner syndrome, Tuberculosis,
Developmental disorders, Psoriasis, Rieger syndrome, Neurological
disorders, Neurodegenerative disorders, Waardenburg syndrome,
Niemann-Pick disease, Myelodysplastic syndrome, Myocardial
infarction, Hypertension, Angiogenesis, Erythropoiesis, Congenital
heart disease, HDR syndrome, Myelodysplastic syndrome, Arthiritis,
Polyglutamine disorders, solid tumors and cancer
1TABLE 1 Listing of particularly preferred genes of the present
invention associated with DNA transcription Database Entry No.
(Genbank a Gene http://www.ncbi.nlm.nih.gov) ELF1 M82882 ETV3
L16464 ETV4 D12765 TAF2C2 Y09321 TCF9 M29204 ZNF121 M99593 ZNF131
U09410 ZNF139 U09848 ZNF154 U20648 ZNF169 U28322 ZNF2 X60152 ZNF204
AF033199 ZNF206 AA206569 ZNF3 X78926 ZNF37A X69115 ZNF44 X16281
ZNF8 M29581 ZK1 NM_005815 ADA NM_000022 ATBF1 ND_006885 ATF3
NM_001674 ZNF255 NM_005774 CBFB NM_001755 CEBPD NM_005195 CTPS
NM_001905 DCTD NM_001921 DR1 NM_001938 ELF3 NM_004433 ELK4
NM_021795 ETV5 NM_004454 KLF4 NM_004235 FOXO1A NM_002015 FLI1
NM_002017 HHEX NM_001529 HIVEP1 NM_002114 HMG2 NM_002129 ID1
NM_002165 ID3 NM_002167 LAF4 NM_002285 ZNFN 1A1 NM_006060 LYL1
NM_005583 MAFG NM_002359 MAZ NM_002383 ODC1 NM_002539 PBX3
NM_006195 POU2AF1 NM_006235 POU2F2 NM_002698 POU3F1 NM_002699 PROP1
NM_006261 RARG NM_000966 RECQL NM_002907 RXRA NM_002957 SP100
NM_003113 ZFP93 NM_004234 MKRN3 NM_005664 ZF133 NM_003434 ZNF157
NM_003446 ZNF173 NM_003449 ZNF189 NM_003452 ZNF207 NM_003457 ZNF262
NM_005095 ZNF264 NM_003417 ZNF74 NM_003426 ZNF91 NM_003430 POU3F2
NM_005604 ZNF84 NM_003428 ERV3 TCF8
[0119]
Sequence CWU 0
0
* * * * *
References