WO2009108917A2 - Markers for improved detection of breast cancer - Google Patents

Markers for improved detection of breast cancer Download PDF

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WO2009108917A2
WO2009108917A2 PCT/US2009/035654 US2009035654W WO2009108917A2 WO 2009108917 A2 WO2009108917 A2 WO 2009108917A2 US 2009035654 W US2009035654 W US 2009035654W WO 2009108917 A2 WO2009108917 A2 WO 2009108917A2
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seq
gene
cancer
methylation
genes
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WO2009108917A3 (en
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James Herman
Nita Ahuja
Kornel E. Scheubel
Leslie Cope
Valerie Deregowski
Wim Van Criekinge
Stephen Baylin
Leander Van Neste
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MDxHealth SA
Johns Hopkins University
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OncoMethylome Sciences SA
Johns Hopkins University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates generally to cancer diagnosis and therapeutics.
  • it relates to the identification of a cell proliferative disorder of breast by determining aberrant DNA methylation patterns of particular genes in breast cancer and pre-cancer.
  • Breast cancer is one of the most significant health concerns. Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer death in women. One in eight women (13% of women) will develop breast cancer during her lifetime. Additionally, a small number of men are diagnosed with or die from breast cancer.
  • tumor suppressor function leads to the initiation and progression of human cancer.
  • Inactivation of tumor suppressor genes can result from both genetic mechanisms such as mutation and epigenetic mechanisms such as DNA hypermethylation (Ponder B.A, 2001; Herman et al., 2003).
  • up-regulation of expression of a number of genes e.g. MAGE-Al
  • Identification of these genes provides insight into the biological processes underlying oncogenesis and is useful for developing new therapeutic and diagnostic modalities.
  • Epigenetic silencing is a second mechanism by which abnormal gene inactivation can occur in cancer.
  • a predominant mode of epigenetic alteration in cancer is gene silencing via CpG island promoter hypermethylation (henceforth called hypermethylation).
  • Hypermethylation acts by recruiting methyl-cytosine-binding proteins and histone deacetylases, which in a coordinated fashion, modify nucleosomes to form transcriptionally repressive chromatin (Busslinger et al., 1983; Nan et al., 1998).
  • Repressive histone marks such as methylation of lysine-9 on histone 3 (H3K9) may initiate and help maintain this state of repression (Jenuwein et ah, 2006; Barski et ah, 2007). This results in the activation of many oncogenes and silencing of tumor suppressors to promote proliferation of abnormal cells.
  • hypomethylation of a gene may lead to aberrant expression of certain antigens in a wide variety of tumors. These epigenetic abnormalities (hyper- and hypomethylation) could cooperate with genetic alterations to effect aberrant gene function that results in cancer.
  • DNA methylation markers have been evaluated as potential genetic markers for detection of cancer because they offer certain advantages when compared to mutation markers: DNA hypermethylation appears to be an early event in the etiology of carcinogenesis and appears to precede apparent malignancy in many cases (Esteller et ah, 2001). The methylation profile is in many cases tissue- and tumor-type specific and is also preserved in purified isolated DNA. In addition, methylation markers may serve for predictive purposes as they often reflect the sensitivity to therapy or duration of patient survival. DNA hypermethylation is also prevalent in the highly aggressive HER2/Neu-positive breast cancers.
  • the present invention is based on the finding that several genes are newly identified as being differentially methylated in breast cancers. This information is useful for breast cancer screening, risk-assessment, prognosis, disease identification, disease staging and identification of therapeutic targets. The identification of new genes that are methylated in breast cancer allows accurate and effective early diagnostic assays, methylation profiling using multiple genes and identification of new targets for therapeutic intervention.
  • the invention provides for a method for identifying breast cancer or its precursor, or predisposition to breast cancer.
  • Epigenetic modification is detected in a test sample containing breast cells or nucleic acids from breast cells.
  • the epigenetic modification is of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK
  • the epigenetic modification may be detected by determining the state of methylation of one or more nucleic acids from a subject.
  • a difference in the state of methylation of one or more nucleic acids in a test sample of the subject compared to the state of methylation of one or more nucleic acids from a subject not having a cellular proliferative disorder of breast tissue is indicative of the test sample containing breast cells that are neoplastic, precursors to neoplastic, or predisposed to neoplasia, or as containing nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
  • the invention also provides for a method for determining the histopathological stage of breast cancer, comprising detecting epigenetic modification of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2;
  • a method is provided of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer.
  • An epigenetically silenced gene is determined in a cell.
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7
  • a polypeptide encoded by the epigenetic silenced gene in the cell is restored by contacting the cell with one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor. Unregulated growth of the cell is thereby reduced or inhibited.
  • reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer is accomplished by introduction of a polynucleotide encoding a polypeptide into the cell.
  • the polypeptide is encoded by said gene.
  • the polypeptide is expressed in the cell thereby restoring expression of the polypeptide in the cell.
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKN
  • One or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor is administered to the patient in sufficient amounts to restore expression of the epigenetic silenced gene in the patient's cancer cells.
  • HDAC histone deacetylase
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from those shown in HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA
  • a polynucleotide encoding a polypeptide is administered to the patient.
  • the polypeptide is encoded by the epigenetic silenced gene.
  • the polypeptide is expressed in the patient's tumor thereby restoring expression of the polypeptide in the cancer.
  • a method for selecting a therapeutic strategy for treating a cancer patient.
  • a gene is identified whose expression in cancer cells of the patient is reactivated by a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, or a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A
  • kits for assessing methylation in a test sample comprises at least the following reagents: a reagent that (a) modifies methylated cytosine residues but not non-methylated cytosine residues, or that (b) modifies non- methylated cytosine residues but not methylated cytosine residues; and a pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp of said gene's transcription start site, said gene being selected from those shown in HOXDl; SLC2A14; NEFH; HOXA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl;
  • the invention provides oligonucleotide primers and/or probes and their sequences for use in the methods and assays of the invention.
  • FIG. 1A-1B Strategy used to identify common targets of mutation and promoter hypermethylation.
  • FIG. IA Large-scale sequencing of breast and colon cancers identified 189 genes cancer genes (CAN genes) as reported by Sj ⁇ blom et al. Candidate hypermethylated genes were identified via expression microarray analysis. Genes without promoter CpG islands were excluded resulting in 36 target genes.
  • FIG. IB Frequency of promoter methylation in the 36 CAN genes that are subject to hypermethylation. X-axis denotes percent methylation. Methylation status was determined in the 6 colorectal cancer cell lines and 11 breast cancer cell lines as indicated in the example section.
  • FIG. 2 Primers used for Methylation-specific PCR of the 36 target genes. (SEQ ID NO: 1305-1448, respectively)
  • FIG. 3. Promoter methylation frequency in primary colon and breast tumors: 18 out of the 36 common target genes methylated in either breast cancer cell lines, colon cancer cell lines or both, showed cancer-specific methylation. Y-axis denotes percent methylation.
  • FIG. 4 Mutation and methylation plot of the 18 genes showing cancer-specific methylation..
  • FIG. 5 Primary breast tumor grade or stage correlation. Y-axis denotes percent methylation for the different tumor stages/grades.
  • FIG. 6A-6B FIG. 6A. Position of the different primers relative to the TSS.
  • FIG. 6B Sequences of the different primer sets, sense (SEQ ID NO: 1273-1280, respectively) and antisense (SEQ ID NO: 1281-1288, respectively) and amplicons. (SEQ ID NO: 1289-1296, respectively)
  • Fig. 7 Methylation frequency SLC2A14, NEFH and TF. Percentage methylation (Y-axis) is shown for each marker (X-axis)
  • microarray screen identifies hypermethylated genes that are re-expressed following treatment with the DNA methyltransferase (DNMT) inhibitor 5-deoxyazacytidine (DAC) but not following treatment with the HDAC I/II inhibitor trichostatin A (TSA) alone.
  • DNMT DNA methyltransferase
  • DAC 5-deoxyazacytidine
  • TSA HDAC I/II inhibitor trichostatin A
  • a further substantial fraction of the cancer gene promoter CpG island DNA hypermethylome is defined through comparison of the hypermethylome genes found by the micro human transcriptome microarray screen and their specificity for certain cell lines such as metastatic cell lines but not for non-metastatic cell lines. Resulting methylation markers are useful in identifying cancers with high metastatic potential and may provide guidance to a preferred method of treatment. For instance, for cancers with high metastatic potential it may be indicated to have radiotherapy treatment following the resection of the cancer.
  • genes include: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34
  • Detection of epigenetic silencing of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of such genes can be used as an indication of cancer or pre-cancer or risk of developing cancer.
  • accession numbers corresponding to the listed genes can be found at the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health website.
  • silencing when used in reference to a gene, means that the gene is not being transcribed, or is being transcribed at a reduced level when compared to the transcription level of the same gene in a corresponding control cell, e.g., a healthy cell, a non-cancerous cell, a non-infected cell.
  • a cell comprising the silenced gene has reduced levels of, or completely lacks, a polypeptide encoded by the gene, i.e. the gene product, such that any function normally attributed to the gene product in the cell is reduced of absent.
  • Assays that recognize only a single marker of the listed markers may be of limited predictive value.
  • a multiplexed analytical approach is particularly suitable for cancer diagnostics, and thus a panel approach may be more consistent with the heterogeneous nature of cancer. Combinations of markers may be applied as desired. Multiplexing in connection to epigenetic silencing of genes has been described in WO05042713 and such methods are applicable in the present invention.
  • the cancer to be identified is preferably a breast cancer.
  • breast cancer There are two major groups of breast cancer: noninvasive carcinoma and invasive carcinoma.
  • the noninvasive carcinomas include lobular carcinoma in situ and ductal carcinoma in situ.
  • breast cancers often grow through the basement membrane and roughly 95% of all breast cancers are infiltrating or invasive carcinomas.
  • the most common type of invasive breast cancer (about 75%) is invasive ductal carcinoma, arising in the milk ducts and spreading through the duct walls.
  • Invasive lobular carcinoma originates in the milk glands and accounts for 10 to 15% of invasive breast cancers.
  • invasive breast cancer Less common types of invasive breast cancer include the following: inflammatory breast cancer, Paget's disease of the nipple, medullary carcinoma, mucinous carcinoma, phyllodes tumor, and tubular carcinoma. Rarely (about 1%), sarcomas (cancer of the connective tissue) develop in the breasts. Individuals may develop one, the other, or a combination of invasive and noninvasive breast cancer.
  • Epigenetic silencing of a gene can be determined by any method known in the art. One method is to determine that a gene which is expressed in normal cells or other control cells is less expressed or not expressed in tumor cells. This method does not, on its own, however, indicate that the silencing is epigenetic, as the mechanism of the silencing could be genetic, for example, by somatic mutation.
  • One method to determine that the silencing is epigenetic is to treat with a reagent, such as DAC (5'-deazacytidine), or with a reagent which changes the histone acetylation status of cellular DNA or any other treatment affecting epigenetic mechanisms present in cells, and observe that the silencing is reversed, i.e., that the expression of the gene is reactivated or restored.
  • a reagent such as DAC (5'-deazacytidine)
  • Another means to detect epigenetic silencing is to determine the presence of methylated CpG dinucleotide motifs in the silenced gene.
  • Methylation of a CpG island at a promoter usually prevents expression of the gene.
  • the islands can surround the 5' region of the coding region of the gene as well as the 3' region of the coding region.
  • CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns. All of these regions can be assessed to determine their methylation status.
  • these reside near the transcription start site and surround the TTS, for example, within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp.
  • RNA can also be assessed using amplification techniques, such as RT-PCR.
  • Sequencing-based methods are an alternative; these methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS).
  • SAGE serial analysis of gene expression
  • MPSS massively parallel signature sequencing
  • Differential display techniques provide another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest.
  • Specific proteins can be assessed using any convenient method including immunoassays and immuno- cytochemistry but are not limited to that. Most such methods will employ antibodies which are specific for the particular protein or protein fragments.
  • the sequences of the mRNA (cDNA) and proteins of the markers of the present invention are known in the art and publicly available.
  • microarray refers broadly to both “DNA microrarray” and “DNA chips”, and encompasses all art-recognized solid supports and methods for affixing nucleic acid molecules thereto or synthesis of nucleic acids thereon.
  • Methylation-sensitive restriction endonucleases can be used to detect methylated CpG dinucleotide motifs. Such endonucleases may either preferentially cleave methylated recognition sites relative to non-methylated recognition sites or preferentially cleave non- methylated relative to methylated recognition sites.
  • Non limiting examples of the former are Aat II, Ace III, Ad I, AcI I, Age I, AIu I, Asc I, Ase 1, AsiS I, Ban I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrV I, BssK 1, BstB I, BstN I, Bs I, CIa I, Eae I, Eag I, Fau I, Fse I, Hha I, mPl I, HinC II, Hpa 11, Npy99 I, HpyCAIV, Kas I, Mbo I, MIu I, MapA 1 1.
  • Msp I Nae I, Nar I, Not 1, PmI I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, SfI I, Sfo I, SgrA I, Sma I SnaB I, Tsc I, Xma I, and Zra I.
  • Non limiting examples of the latter are Ace II, Ava I, BssH II, BstU I, Hpa II, Not I and Mho I.
  • chemical reagents can be used which selectively modify either the methylated or non-methylated form of CpG dinucleotide motifs. Modified products can be detected directly, or after a further reaction which creates products which are easily distinguishable. Means which detect altered size and/or charge can be used to detect modified products, including but not limited to electrophoresis, chromatography, and mass spectrometry. Examples of such chemical reagents for selective modification include hydrazine and bisulfite ions. Hydrazine-modified DNA can be treated with piperidine to cleave it. Bisulfite ion-treated DNA can be treated with alkali. Other means for detection which are reliant on specific sequences can be used, including but not limited to hybridization, amplification, sequencing, and ligase chain reaction. Combinations of such techniques can be used as is desired.
  • the principle behind mass spectrometry is the ionizing of nucleic acids and separating them according to their mass to charge ratio. Similar to electrophoresis, one can use mass spectrometry to detect a specific nucleic acid that was created in an experiment to determine methylation. See Tost, J. et al. Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry.
  • chromatography high performance liquid chromatography
  • DNA is first treated with sodium bisulfite, which converts an unmethylated cytosine to uracil, while methylated cytosine residues remain unaffected.
  • One may amplify the region containing potential methylation sites via PCR and separate the products via denaturing high performance liquid chromatography (DHPLC).
  • DHPLC has the resolution capabilities to distinguish between methylated (containing cytosine) and unmethylated (containing uracil) DNA sequences.
  • Deng, D. et al. describes simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography.
  • Hybridization is a technique for detecting specific nucleic acid sequences that is based on the annealing of two complementary nucleic acid strands to form a double-stranded molecule.
  • One example of the use of hybridization is a microarray assay to determine the methylation status of DNA. After sodium bisulfite treatment of DNA, which converts an unmethylated cytosine to uracil while methylated cytosine residues remain unaffected, oligonucleotides complementary to potential methylation sites can hybridize to the bisulfite-treated DNA.
  • the oligonucleotides are designed to be complimentary to either sequence containing uracil or sequence containing cytosine at the original CpG islands, representing unmethylated and methylated DNA, respectively.
  • Computer-based microarray technology can determine which oligonucleotides hybridize with the DNA sequence and one can deduce the methylation status of the DNA.
  • An additional method of determining the results after sodium bisulfite treatment would be to sequence the DNA to directly observe any bisulfite-modifications.
  • Pyrosequencing technology is a method of sequencing-by-synthesis in real time.
  • PPi pyrophosphate
  • dNTP deoxynucleotide
  • This method presents a DNA template-primer complex with a dNTP in the presence of an exonuclease-deficient Klenow DNA polymerase.
  • the four nucleotides are sequentially added to the reaction mix in a predetermined order. If the nucleotide is complementary to the template base and thus incorporated, PPi is released.
  • the PPi and other reagents are used as a substrate in a luciferase reaction producing visible light that is detected by either a luminometer or a charge-coupled device.
  • the light produced is proportional to the number of nucleotides added to the DNA primer and results in a peak indicating the number and type of nucleotide present in the form of a pyrogram. Pyrosequencing can exploit the sequence differences that arise following sodium bisulfite-conversion of DNA.
  • present methods use amplification techniques in a reaction for creating distinguishable products.
  • Multiple amplification techniques are known. Some of these techniques employ PCR.
  • Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), transcription amplification (Kwoh et al. 1989; WO88/10315), selective amplification of target polynucleotide sequences (US Patent No.
  • Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to PCR primer design.
  • the primers do not themselves "cover” or hybridize to any potential sites of DNA methylation; sequence variation at sites of differential methylation are located between the two primers.
  • Such primers are used in bisulphite genomic sequencing, COBRA, Ms-SNuPE.
  • the primers are designed to anneal specifically with either the methylated or unmethylated version of the converted sequence.
  • the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations.
  • additional nucleotide residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After primer hybridization, an amplification reaction can be performed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not. For example, bisulfite ions convert non- methylated cytosine bases to uracil bases. Uracil bases hybridize to adenine bases under hybridization conditions.
  • an oligonucleotide primer which comprises adenine bases in place of guanine bases would hybridize to the bisulfite-modified DNA, whereas an oligonucleotide primer containing the guanine bases would hybridize to the non-converted (initial methylated) cytosine residues in the modified DNA.
  • Amplification using a DNA polymerase and a second primer yield amplification products which can be readily observed. This method is known as MSP (Methylation Specific PCR; Patent Nos 5,786,146; 6,017,704; 6,200,756).
  • Preferred primers and primer sets for assessing the methylation status of the concerned gene by way of MSP are provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
  • the amplification products can be optionally hybridized to specific oligonucleotide probes which may also be specific for certain products. Alternatively, oligonucleotide probes can be used which will hybridize to amplification products from both modified and non- modified DNA.
  • present invention provides for a method for detecting breast cancer or its precursor, or predisposition to breast cancer in a test sample containing breast cells or nucleic acids from breast cells comprising:
  • CTAGlA CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27;
  • NEURL NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA;
  • PPPl Rl 4A PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1;
  • TIMP3 TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl;
  • Modified and non-modified DNA can be distinguished with use of oligonucleotide probes which may also be specific for certain products. Such probes can be hybridized directly to modified DNA or to amplification products of modified DNA. Probes for assessing the methylation status of the concerned gene will specifically hybridize to the converted sequence but not to the corresponding non converted sequence. Probes are designed to anneal specifically with the converted sequence representing either the methylated or unmethylated version of the DNA. Preferred converted sequences are provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495- 1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
  • Preferred probes anneal specifically with the converted sequence representing the methylated version of the DNA, in particular those sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501- 1503), and Figure 6B (SEQ ID NO: 1289-1304).
  • Oligonucleotide probes can be labeled using detection systems known in the art. These include but are not limited to fluorescent moieties, radioisotope labeled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.
  • Real time chemistry allows for the detection of PCR amplification during the early phases of the reactions, and makes quantitation of DNA and RNA easier and more precise.
  • a few variants of real-time PCR are well known. They include Taqman® (Roche Molecular Systems), Molecular Beacons®, Amplifluor® (Chemicon International) and Scorpion® DzyNA®, PlexorTM (Promega) etc.
  • the TaqMan® system and Molecular Beacon® system have separate probes labeled with a fluorophore and a fuorescence quencher.
  • the labeled probe in the form of a hairpin structure is linked to the primer.
  • Quantitation in real time format may be on an absolute basis, or it may be relative to a methylated DNA standard or relative to an unmethylated DNA standard.
  • the absolute copy number of the methylated marker gene can be determined; or the methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene with a known methylation (e.g. ⁇ -actin), or by using the ratio between the methylated marker and the sum of the methylated and the non- methylated marker.
  • identification of methylated CpG dinucleotides may utilize the ability of the MBD domain of the McCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999). Restriction enconuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Variants of this method have been described and may be used in present methods of the invention.
  • Test samples for diagnostic, prognostic, or personalized medicine uses can be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin embedded colon, rectum, small intestinal, gastric, esophageal, bone marrow, breast, ovary, prostate, kidney, lung, brain or other organ tissues, from lymph nodes, or from a body fluid such as blood, serum, lymph, cerebrospinal fluid, saliva, sputum, bronchial -lavage fluid, ductal fluids stool, urine, or semen.
  • a test sample obtainable from such specimens or fluids includes detached tumor cells or free nucleic acids that are released from dead or damaged tumor cells.
  • Nucleic acids include RNA, genomic DNA, mitochondrial DNA, single or double stranded, and protein-associated nucleic acids. Any nucleic acid specimen in purified or non-purified form obtained from such specimen cell can be utilized as the starting nucleic acid or acids. Particular suitable for breast cancer assays are blood derived samples such as serum and plasma. Alternatively, fluid within the mammary glands is used. Such fluid can be obtained from the breast e.g. by nipple aspiration of the milk ducts or by ductal lavage of at least one breast milk duct or from spontaneous nipple discharge. Administration of oxytocin may be used to stimulate expression of mammary fluid from a nipple of the patient.
  • Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA, and leads to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions.
  • Analysis of RARbeta2 and RASSFlA methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women (Skvortsova et ⁇ /. 3 2006).
  • primers and probes useful in MSP carried out on the gene selected from HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH
  • primers specifically hybridize to the sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
  • Preferred primers comprise, consist essentially of, or consist of the sequences provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
  • probes specifically hybridize to the sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
  • Preferred probes (5' - 3') may comprise, consist essentially of or consist of the sequences represented by SEQ ID NO. 1485-1494.
  • Alternative fluorescent donor and acceptor moieties/FRET pairs may be utilized as appropriate.
  • the primers and probes may include modified oligonucleotides and other appending groups and labels provided that the functionality as a primer and/or probe in the methods of the invention is not compromised.
  • an enrichment step prior to the amplification step may be appropriate.
  • the nested MSP approach (US 7,214,485) for instance may provide a solution.
  • the generation and amplification of a DNA library before testing for methylation of any specific gene may be required.
  • Suitable methods on whole genome amplification and libraries generation for such amplification e.g., Methylplex and Enzyplex technology, Rubicon Genomics
  • WO2005/090507 regards library generation/amplification methods that require either bisulphite conversion or non-bisulphite based application.
  • Bisulphite treatment may occur before or after library construction and may require the use of adaptors resistant to bisulphite conversion.
  • Meth- DOP-PCR (Di Vinci et al, 2006), a modified degenerate oligonucleotide-primed PCR amplification (DOP-PCR) that is combined with MSP, provides another suitable method for specific detection of methylation in small amount of DNA. Improved management of patient care may require these existing methods and techniques to supplement the methods of the invention.
  • CA 15-3 CA 27.29, CEA, carcinoembryonic antigen (CEA), estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-I).
  • CEA carcinoembryonic antigen
  • ER estrogen receptor
  • PgR progesterone receptor
  • HER2 human epidermal growth factor receptor 2
  • uPA urokinase plasminogen activator
  • PAI-I plasminogen activator inhibitor 1
  • BRACAnafysis® assesses a woman's risk of developing breast or ovarian cancer based on detection of mutations in the BRCAl and BRCA2 genes.
  • HER2/neu gene Approximately 25-30 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression of this receptor is associated with highly aggressive breast cancer that requires special treatment, increased disease recurrence and worse prognosis. DNA hypermethylation is prevalent in these highly aggressive HER2/Neu-positive breast cancers. To aid prognostication, it may be necessary to combine the methods of the invention with this established marker for breast cancer prognosis.
  • stage of a cancer is a descriptor (usually numbers I to IV) of how much the cancer has spread.
  • the stage takes into account the size of a tumor, how deep it has penetrated, whether it has invaded adjacent organs, if and how many lymph nodes it has metastasized to, and whether it has spread to distant organs. Staging of cancer is important because the stage at diagnosis is the biggest predictor of survival, and treatments are often changed based on the stage.
  • the "grade” of a cancer refers to cell appearance (differentiation) and DNA make up.
  • the invention also provides for a method for determining the stage of cancer comprising determining epigenetic silencing of a gene in cancers, cancer precursors, and pre-cancers.
  • the genes include: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK
  • SYNEl and COL7A1 are preferentially methylated in advanced tumors.
  • PTPRD, SYNEl, and EVL are preferentially hypermethylated in high grade tumors.
  • These markers are preferred markers for determining the stage and/or grade of the breast cancer. Since grade is a strong predictor of local recurrence and metastasis, such markers are useful in predicting aggressive clinical behavior of the cancer and prognosis.
  • Testing can be performed diagnostically or in conjunction with a therapeutic regimen. Testing can be used to monitor efficacy of a therapeutic regimen, whether a chemotherapeutic agent or a biological agent, such as a polynucleotide. Testing can also be used to determine what therapeutic or preventive regimen to employ on a patient. Moreover, testing can be used to stratify patients into groups for testing agents and determining their efficacy on various groups of patients. [69] According to another aspect of the invention a method is provided of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer. An epigentically silenced gene is determined in a cell.
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A
  • a polypeptide encoded by the epigenetic silenced gene in the cell is restored by contacting the cell with one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor. Unregulated growth of the cell is thereby reduced or inhibited.
  • a CpG dinucleotide demethylating agent a DNA methyltransferase inhibitor
  • HDAC histone deacetylase
  • Another aspect of the invention is a method of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer.
  • An epigenetic silenced gene is determined in the cell.
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK
  • Yet another aspect of the invention is a method of treating a cancer patient.
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDK
  • One or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor is administered to the patient in sufficient amounts to restore expression of the epigenetic silenced gene in the patient's cancer cells.
  • HDAC histone deacetylase
  • Yet another aspect of the invention is a method of treating a cancer patient.
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from those shown in HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA
  • a polynucleotide encoding a polypeptide is administered to the patient.
  • the polypeptide is encoded by the epigenetic silenced gene.
  • the polypeptide is expressed in the patient's tumor thereby restoring expression of the polypeptide in the cancer.
  • a method for selecting a therapeutic strategy for treating a cancer patient.
  • a gene is identified whose expression in cancer cells of the patient is reactivated by a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, or a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7
  • Demethylating agents can be contacted with cells in vitro or in vivo for the purpose of restoring normal gene expression to the cell.
  • Suitable demethylating agents include, but are not limited to 5-aza-2'-deoxycytidine, 5-aza-cytidine, Zebularine, procaine, and L- ethionine. This reaction may be used for diagnosis, for determining predisposition, and for determining suitable therapeutic regimes.
  • expression or methylation can be tested of a gene selected from HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl
  • kits according to the present invention are assemblages of reagents for testing methylation. They are typically in a package which contains all elements, optionally including instructions. The package may be divided so that components are not mixed until desired. Components may be in different physical states. For example, some components may be lyophilized and some in aqueous solution. Some may be frozen. Individual components may be separately packaged within the kit.
  • the kit may contain reagents, as described above for differentially modifying methylated and non-methylated cytosine residues.
  • the kit will contain oligonucleotide primers which specifically hybridize to regions within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp kb of the transcription start sites of the genes/markers: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl
  • the kit will contain both a forward and a reverse primer for a single gene or marker. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • the kit may optionally contain oligonucleotide probes.
  • the probes may be specific for sequences containing modified methylated residues or for sequences containing non-methylated residues.
  • the kit may optionally contain reagents for modifying methylated cytosine residues.
  • the kit may also contain components for performing amplification, such as a DNA polymerase and deoxyribonucleotides. Means of detection may also be provided in the kit, including detectable labels on primers or probes.
  • Kits may also contain reagents for detecting gene expression for one of the markers of the present invention. Such reagents may include probes, primers, or antibodies, for example. In the case of enzymes or ligands, substrates or binding partners may be sued to assess the presence of the marker.
  • the invention provides for oligonucleotide primers and/or probes and their sequences for use in the methods and assays of the invention.
  • Preferred primers and their sequences bind to at least one of the polynucleotide sequences listed in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304) or to the complement sequence thereof.
  • Preferred probes and their sequences bind to at least one of the polynucleotide sequences listed in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304) or to the complement sequence thereof.
  • Preferred probes (5' - 3') may comprise, consist essentially of or consist of the sequences represented by SEQ ID NO. 1485-1494, or the complement sequence thereof.
  • the invention also provides for an isolated polynucleotide which consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-1503.
  • ACP5_55536 54 ACP5 TTTTGTATTTGCGGTCGGT AAAAACTCCTAAATATCCGCGA
  • ACP5_55540 54 ACP5 CGGTTTATTTTATAGATGCGGAG AAAAACGAACGCCTCGACC
  • ACTN2_55535 88 ACTN2 GTGTTTTGTTTGTTTTCGGAC TAACCGCGTAACCTAACGACT
  • ADRA2A_57683 150 ADRA2A GGTAGAGTTCGCGTTTTAGTTTC TCGAAAATCCTATATCTATCGCC
  • ADRA2A_57684 150 ADRA2A GTTTTTGTTTTAATTCGCGTTG ATTCTCCTAAACGTCCGCCT
  • ARMCX2_13030 9823 ARMCX2 TTTTACGTGATTAGGAGTTGACG ATTAAACGCACGACAACGAA
  • ATXN1_56027 6310 ATXN 1 TACGGGGATAGATTTGGGAGC ATAAACCGACAAAAACCTCGATA
  • BIK_22360 638 BIK TTTTTGGAGTTTCGGTTTTTAC CACGAATAACCTCCGTTCG
  • CCND2_1 894 CCN D2 GAGTTTCGGGGTTGTTTTATTC CCAACTTACGTCACCGCTT
  • CCND2_25209 894 CCN D2 GAAGGTAGCGTTTTTCGATG AAATAAACCCGATCCGCAA
  • CDKN2A_9696 1029 CDKN2A TTTAAGTTTTTAGGGCGTCGTT AAATAACGCTTCGATTCTCCG
  • CFTR_55912 1080 CFTR TCGAGAGATTATGTAGAGGTCGTT CCTCCTCTTTCGTAAACACGTA
  • CFTR_55913 1080 CFTR AGTTTTTCGGGGAGTCGGT GCAAATAAACGACAATCGC
  • CNIH3_55879 1491 11 CNIH3 TTATTAAATCGTTGAGGAGAGACGA GCTAAACACTATCCGTTTACGCC
  • CNN3 1266 CNN3 GCCTAAACCTTCGAAAATTAACCG
  • COL7A1_55842 1294 COL7A1 CGGTTATTAGAAGTCGTAGCGT GAACCTACCAAAACCACGAAA
  • COL7A1_55846 1294 COL7A1 GGGTGGTACGGTGTAGTGTTTC TAACAAAAACGCCCCGAAT
  • COL9A3_161 15 1299 COL9A3 TAGGTTTACGGGGGTATTTGC TCCTAACCGAACTTCCCGAAC
  • COL9A3_161 18 1299 COL9A3 TCGCGTAGTGTGTTTTTCGTT ATAAATCCTCGCTAACCCGAAT
  • CTAG2 246100 CTAG 1 A GTGAGAATCGGTTACGTGTTTC GAAATCCTCAAAACGCCTACG
  • CYP1A1_23831 1543 CYP1A1 TATTCGTGGTGGTGTTGAGC AAAATATAAAAATCGAACCGACC
  • ESR2_55653 2100 ESR2 TTTTTAGTTTTGGGGACGC CTATAAAATCCAACGCCCG
  • FAM20B_55630 9917 FAM20B ATATAGGGATACGCGGTTCGAG CCGTCCCCGAATAACTACG
  • FAS_18139 355 FAS GGAGTTGTTTCGTTTGTTTAGC AACCCGAATACTCAAAACGC
  • FBLN1_54935 2192 FBLN 1 ATTTCGAGTTTTCGTGGTTTC CGACCTTACGCTACTAACGACC
  • GALE_new2 2582 GALE GTTATTGGGATTTGGCGTC GCTATAACGAAAACTACGCAACG
  • GJB2_22306 2706 GJB2 TGCGGAGTATAGAGGATAACGA AACGAAAACGACTAAAAATCGAAA
  • GPNMB_52603 10457 GPNMB TTAATTTTTAGTTTTTCGATTGCG GTACTACGCGCTAACACCGA
  • GPNMB_52605 10457 GPNMB GAAGCGGTTAAAGGCGTAG TAAAAATTAAAACGCGACCG
  • GREM1_29777 26585 GREM1 GAATTTGGTACGATTTTACGGAG ATCTAAACTTTCCCTATCGACCG
  • HIST1 H3G_57040 8355 HIST1 H3G CGATTTTTCGTATTAGGCGTTG AAAACCTCATCGCTACCGTC
  • HIST1 H3G_57042 8355 HIST1 H3G CGGGTGAAAGTAGGCGGTT AAACATTCAACTCGCTCGC
  • HOXA10_57002 3206 HOXA10 TTTATTCGGTAAGATCGGGG AATCTAAATCCCGAAACGCA
  • HOXA5_1 3202 HOXA5 GTAGTTCGGGTTATTTGGATAGC AAAAATACTATAAACGCACAAACGA
  • ICAM1 3383 ICAM1 TAAAGACGTTTTCGCGGTTAAGGTC ACCACGTCCGAAAAAATCGACG
  • ID4_56952 3400 ID4 GCGAGTAGGGTTTAGGCGTT AAAACTACGAAAATATACGACCGA
  • INHBB_56906 3625 INHBB GTATTTCGTGTCGCGGTTC CCGAATACAATACCCTCGCT
  • ITIH5_56887 80760 ITIH5 CGTTGTAAAGCGTGTTTCGT CTACGCCTCTTCCTACGACC
  • KCNG3_56878 170850 KCNG3 GAGGGTTGCGTATATCGAGG ACGAAAAACTATTCCGCCC
  • KRTCAP3_56842 200634 KRTCAP3 GTTGATGCGTGTGGGTTTC AAAATTAACCACGTACCGCAA
  • LZTFL1_56661 54585 LZTFL1 GTCGGTTATTTATTATGGCGGT ACGATCCCAAACTTAACGAAA
  • MAL 1 4118 MAL TTCGGGTTTTTTTGTTTTTAATTC GAAAACCATAACGACGTACTAACG 254 MAL_56656 4118 MAL GTGGCGGTGGTTTAGTTTC TTCATTTTTCCGCTAAATACGTT
  • NAGS_57828 162417 NAGS GGGTGTCGAGTTTTAGGTTTTC TAAATATCCCGCTACACCAACGA
  • NDP_1 4693 NDP TTTCGTGGAAGGGAGTCGAG CGCACTAACAAATAAAAATACCACG
  • NEF3_13215 4741 NEF3 TGAGTTATACGTTGGATTCGTTG AAAAACCGCTAACGCGACT
  • NPPB_13212 4879 NPPB GGAGTACGGGGTGATTTATAGC CGACGAACAAATACTACGCTACG
  • NPTX2_57773 4885 NPTX2 TTTTAGTTTGTGACGTTCGCGTT TAAAACTCTCGAAAACCTCGACT
  • NPTX2_57779 4885 NPTX2 GCGTCGTTTTGTATGGGTATC CCCGATAACCGCTTCGTAT
  • OGDHL_19631 55753 OGDHL GTTTTAGTTTCGTTTTGCGGTT GCTCCTAACGCTATCCACG
  • PIK3CA_66163 5290 PIK3CA AAAAATAGGGGCGACGGAG GACACCGAAACTACCGCTT
  • PIK3CA_66165 5290 PIK3CA ATAGAGTTATCGCGGTCGGG CTCTAACTACCGCCTCGCTC
  • RARRES1_57581 5918 RARRES 1 GCGAAATCGTAGGGGAAAC ATAAAAACGCCTCCCCGAAA
  • RASSF1_8476 11 186 RASSF1 TCGTTTTTAGGAATGATTTTATCG CACTCTTATACGCTTACCCGAAC
  • RASSF1_8480 11 186 RASSF1 GTTTGCGAGTTAGCGAGGTTC TAACCTAAAACAACACGACGA
  • RPRC1_56336 55700 RPRC1 TTTTTATTTAGTTTTTAGGCGTTGC ACCGAACGAACTCCACGAC
  • SESN1_29753 27244 SESN 1 TTTTAGATAGGGCGGGTTTTC AAAAACGACTCACGATACGCAA
  • SESN1_29754 27244 SESN 1 ATTAGGACGAGGTATTTGGGC AAAAACAACGTAACCTCCGTAT
  • SFN 2810 SFN TGGTAGTTTTTATGAAAGGCGTC CCTCTAACCGCCCACCACG
  • TIMP3 7078 TIMP3 GCGTCGGAGGTTAAGGTTGTT CTCTCCAAAATTACCGTACGCG
  • TNFRSF11 B_30721 4982 TNFRSF11 B TATCGGGTTGAGGAATAAGGC AAAAACTAACCGCTAACGAAA
  • TNFRSF11 B_30722 4982 TNFRSF11 B TGGTTTAGGGATTTATTACGAGC AACTACGAAAACGCACCGAAA
  • ACTN2_55534 ACTN2 GCGGCGCGGTTATTAAGTCGCGCGGTAGTTGTTCGTAGTCGGAGTTGGT GTTTCGTTCGAGATTTAGCGTTTAGGCGTGTCGT
  • ACTN2_55535 ACTN2 GTGTTTTGTTTGTTTTCGGACGTGTTTTTTTTTTTAAGGGGTTTTCGGGGT
  • ADAM23_2 8745 ADAM23 GAAGGACGAGAAGTAGGCGGTAGGGCGGCGTGCGGGTCGGGGCGTTG
  • ADAM23_A 8745 ADAM23 GAGGTTTTAAGTTGGCGGAGCGGCGAGGATTTTTGGATTTTTTTGCGTTT
  • ADAMTS15_56144 170689 ADAMTS 15 TAGTAGAAGTATGGCGTCGGGTAGCGTATCGTCGCGTTGTGGGAAGGG
  • ADRA2A_57683 150 ADRA2A GGTAGAGTTCGCGTTTTAGTTTCGGGTCGGGTCGGGTTAGAATCGTAGC
  • ADRA2A_57684 150 ADRA2A GTTTTTGTTTTAATTCGCGTTGTCGTCGGATTTCGGTTTATTTAGTAGCGT
  • APC(2) 324 APC TTATATGTCGGTTACGTGCGTTTATATTTAGTTAATCGGCGGGTTTTCGAC GGGAATGGGGAGCGTTTTGGTTC
  • APC2 10297 APC2 GTCGTTTGTTTAGGTTCGGATCGGGTTTTGTTCGTTTCGGAGTTTTTGTT CGCGTCGCGGAGATTTCGGAGTTCGCGCGTTTCGAGGTTATTTCGGGTC
  • APC2_56103 10297 APC2 TCGGATGGTGAAGTTCGTGAGTGGGTGTGTGCGTAGGATCGGTTGTAGA AACGTTGACGTTTAGTTTATCGGGATTTAGTTT
  • AQP5_56090 362 AQP5 TCGGGATCGAGTTTCGTTTTTTAGGGAGTTCGGGGCGTACGGTATCGAG GAGAGCGCGGGAGTTAATTTGGGCGTATTATGCGTAGGG
  • ARMCX2_13030 9823 ARMCX2 TTTTACGTGATTAGGAGTTGACGTGGGTAAAGGTATTTAAAGTTTTGATC
  • ARMCX2_13032 9823 ARMCX2 TGGCGATGTAGTTTTTACGTGATTAGGAGTTGACGTGGGTAAAGGTATTT
  • TCGTAAGAGGAGG ATP2A2_23355 488 ATP2A GAGGGTTCGAGAAGCGAAGAGGTTTAGGGAAGGCGAGGCGAGGATCGT
  • ATXN1_56027 6310 ATXN 1 TACGGGGAT AGATTTGGGAGCGTTGGGCGGGGAGTAGTTTAGTTTTGTG
  • ATXN1_56032 6310 ATXN 1 GGAGTAGTTGGTTGTCGTCGTCGTAGTTTAAGGATTCGATTTTATGGGGG TGGGGGGTATTTTTTGGCGGGCGGTTCGGCGAGTCGTATAGAGTACGG
  • AXIN2_56020 8313 AXIN2 GAGAGATAGAGAGATTACGTCGATTGTTGAGAGGAATTGGAAGAAGAAA
  • BACH1_56015 571 BACH1 TTTTGTAATTTTTCGCGTGGGTTTTCGGTCGCGGCGATTTTTGTTTCGCG
  • BACH1 56017 571 BACH1 TTTTGTGGGGTTAGCGTTCGTTTTTTTTTTTTTGTTGTTCGCGGGTATTCGG GTACGCGGCGATTCGGTTGAATTAGGGCGTTT
  • BEX1 55859 BEX1 GAGTATTAGTTAATTGGTCGTCGGTTCGTGGGGGTTGGTGAGAAGGAGG GTGAGTTTGGCGGTGACGTACGGTTTTTACGTGATCG
  • BEX1_12842 55859 BEX1 TCGGGGTTTTTATTTGGTTCGTTTTTTTTCGGGTCGGATGTTAGTTCGTC GAGCGTAGGGTAGCGGGGAGTTGGTAGCGAGATACGAGTGACGATT
  • BEX1_12850 55859 BEX1 TTAGTT AATTGGTCGTCGGTTCGTGGGGGTTGGTGAGAAGGAGGGTGAG TTTGGCGGTGACGTACGGTTTTTACGTGATCGGGAGTTGTAGAGT
  • BHMT2_55998 23743 BHMT2 TTCGTTTAAGTGTTTTGTCGAGGTTTTAGGGTTTGGTTAGGGATGGACGT CGGGTGTGAACGGAGTCGTTCGTAGTTTAGCGGTT
  • BIK_17885 638 BIK TTTAGAGTTCGGAGTCGGGTGTTCGGAAGTCGTATTGGAGGATTGTGCG
  • BIK_22360 638 BIK TTTTTGGAGTTTCGGTTTTTACGTGGGTAAAATGATCGTGAAAAAAAGTAT CGAGGAGTATTTTGAGTTCGAACGGAGGTTATTCGTG
  • BRCA1_JH 672 BRCA1 TCGTGGTAACGGAAAAGCGCGGGAATTATAGATAAATTAAAATTGCGATT GCGCGCGGCGTGAGTTCGTTGAGATTT
  • CALCA_2 796 CALCA CGTTTTTATAGGGTTTTGGTTGGACGTCGTCGTCGTCGTTGTTATCGTTT
  • CCK_13382 885 CCK ATTGCGAGGGTTTTTAATGCGGTTGAGAAGAAAGTGAAGATTTCGATTTT TTTTTTTTTTCGAAAGAGTCGATAT AGGTATGAAGATTAGCG
  • CCND2_1 894 CCND2 GAGTTTCGGGGTTGTTTTATTCGTATCGGTTTTTTTTTTAAAATTGGTTTC GTTTTTTTTTGTTCGTTTTTTTTCGTTTTGAAGCGGTGACGTAAGTTGG
  • CCND2_25209 894 CCN D2 GAAGGTAGCGTTTTTCGATGGTGAGTAGGTTTTGTAGGACGCGGTCGTT TCGGAGTAGGTTGCGGTTTCGTACGGTTTTGCGGATCGGGTTTATTT
  • CD34_55940 947 CD34 GCGGTATTTTGGGTTTTGCGCGCGCGTTTTTGCGGATTAGTATTTTTTTC GCGCGGTTTAGAGAGACGTATCGAGTGGAAGATATTA
  • CDKN1A_24717 1026 CDKN1A GGGAAATGTGTTTAGCGTATTAACGTAGGCGAGGGATTGGGGGAGGAG GGAAGTGTTTTTTTGTAGTACGCGAGGTTTCGGGATCGGTTGGTTT
  • CDKN2A_8472 1029 CDKN2A GTTTTGGCGAGGGTTGTTTTCGGTTGGTGTTTTCGGGGGAGATTT AATTT GGGGCGATTTTAGGGGTGTTATATTCGTTAAGTGTTCGGAGTTAATAGT
  • CDKN2A_9696 1029 CDKN2A TTTAAGTTTTTAGGGCGTCGTTAGGAGGAGGTTTGTGATTATAAATTTTTT
  • CDO1_55929 1036 CD01 GTTTACGCGATTTTTGGGACGTCGGAGATAACGGGGTTTTTGGGAAGGC
  • CFTR_55912 1080 CFTR TCGAGAGATTATGTAGAGGTCGTTTTTGGAAAAGGTTAGCGTTGTTTTTA
  • CFTR_55913 1080 CFTR AGTTTTTCGGGGAGTCGGTTTTTTCGTCGGTGGTTTTTTTTGTTTTTTAGC
  • CHL1_55888 10752 CHL1 GAATCGAGTGAAATTATCGGGGAGGGGGTGGGGGGCGTTTTTTTTAAAT
  • CNIH3_55879 1491 11 CNIH3 TTATTAAATCGTTGAGGAGAGACGAGCGTTTCGTTTCGGATTTTTTCGCG
  • CNN3 1266 CNN3 GTTTTCGCGGTTTTTTAATTGGTCGTATCGTTTTTCGGCGTAGAGGCGGG
  • COL7A1_55842 1294 COL7A1 CGGTTATTAGAAGTCGTAGCGTTATTTTAGGTAGTAAAAGTCGTTAGTTA
  • COL7A1_55846 1294 COL7A1 GGGTGGTACGGTGTAGTGTTTCGGGTCGGGTTTTTTTTTGCGGTGTTTAT TTTTTTTTTTGTGTTGGGTTCGTATTCGGGGCGTTTTTGTTA
  • COL9A3_161 15 1299 COL9A3 TAGGTTTACGGGGGTATTTGCGTTTTTTAATGAGTTTTTTTCGTTTTAGAG
  • COL9A3_161 18 1299 COL9A3 TCGCGTAGTGTGTTTTTCGTTTTTTTTCGGCGGCGGGAATAAAGGTTTTT
  • CRIP1J 1396 CRIP1 GTCGTTTTAGGGATTTAGCGTTTTCGGTTTTTTTGAGCGGTTTTTAGTTTC
  • CSPG2_23363 1462 CSPG2 TATTGTAGCGTTGCGCGATTGGGTTCGGCGTTGTTTAGGCGGGTTATATA
  • CST6_17991 1474 CST6 TTAGTTTTAGGTCGCGGGGGCGTATCGCGGGCGTCGGGCGGGGCGGTT
  • CTAG2 246100 CTAG1A GTGAGAATCGGTTACGTGTTTCGGGGTTTATTCGGGGTTTTTTAGGGTCG
  • CYP1A1_23831 1543 CYP 1A1 TATTCGTGGTGGTGTTGAGCGGTTTGGATATTATTCGGTAGGTTTTGGTG CGGTAGGGCGATGATTTTAAGGGTCGGTTCGATTTTTATATTTT
  • CYP24A1_55769 1591 CYP24A1 AATTACGGTCGTCGTTGTCGGTTTTTGTTCGTCGGGGGAGGGCGGGGA GGCGCGTTCGAAGTATATTCGGTGAATTTCGGGTTTCGTATGATTTTT
  • DNAJA4_8894 55466 DNAJA4 TTTTTAGTTTTATTTTTCGGCGTAGGGTTTCGGTTAATATAGTTTTTTAGGT
  • EPB41L3_19072 23136 EPB41L3 GCGTGGGTTTTCGTCGTAGTTTCGCGGAGTTTCGGTGTTTTTTGTAATAG
  • EREG_18104 2069 EREG TTTTCGGGTTTTAACGGGGTGAGGTTAAGAGTGTTTAGAGTTTTATAATT GGTTCGAGGGAGGAGCGGTAGGAGGATCGTTA
  • ESR2_55651 2100 ESR2 GATTGTAATTTGAGCGCGGTTTTTTTAGTAGAGTAAGTATATTTATCGGAT
  • ESR2_55653 2100 ESR2 TTTTTAGTTTTGGGGACGCGGTGTAGAAGTGTGAGGGCGTTCGGTTTTTA
  • ESR2_55656 2100 ESR2 TGAGTTGTAGGAGGTGCGTTCGTTTTTTTTAATAGGTGGCGGCGGGGCG
  • ESR2_55657 2100 ESR2 TTTTCGTTAGGAGGTAGTTGTAAGCGCGGAGGTTGCGAGAAATAATTGTT
  • FAM20B_55630 9917 FAM20B ATATAGGGATACGCGGTTCGAGCGGGTTTTTTAGGAAGTTTCGGGTTTTC GTTCGGGATTCGTTCGTAGTTATTCGGGGACGG
  • FAM84A_55594 151354 FAM84A CGTAGATTTTCGTTTTTGGTTTTCGTTTCGTTATTTTATTGATGGGTAATTA ATTGGATCGTATTATTTATTTTAATTATAGCGAGTTGTTTATAGGGGATTC
  • FAM84A_55600 151354 FAM84A GTCGGTTAGTTTACGTGTTTTTATCGGAGTTATTTTTTTTAGTCGAGTTTTT CGTGTTTTTTTAGGGGTACGAGGGGCGGAGAAGTCGTTATAG
  • FAM84A_55624 151354 FAM84A CGGAGTAGTCGGTTTTAGGGTTCGTTTTAGGACGCGTTTATTGGTTGGG GCGGGTGTTCGAGTTGTGTTAATTTTCGTCG
  • FBLN1_54935 2192 FBLN 1 ATTTCGAGTTTTCGTGGTTTCGAAATTCGTAGGGAGCGCGAGGTCGAGG
  • FBLN2_13328 2199 FBLN2 TAGAGCGGAGGAAGTTGCGGATTTGGGGTGGGGGAATTCGTTCGCGGA TTTTTGGTTTTTATTTCGCGTCGGTTTTTGTGTTCGTATTTG
  • GADD45G_57135 10912 GADD45G TATATTAGAAAGCGGGTGTCGGTTAATAGGCGCGTAGTTTCGCGTTAGTT
  • GALE_new2 2582 GALE GTTATTGGGATTTGGCGTCGGCGGCGTTTTTCGCGGGGTGGTATCGTTT
  • GDA_57108 9615 GDA GTATCGGTAATCGTTCGGGTAAGCGGGGGTAGGATAAGGTCGGAGTTTG TGTTCGTTCGGTAGTCGTTCGTAGTTGTAGAGAGTTTCGTTG
  • GDF10_57718 2662 GDF10 CGGGGATATGAGTTATGGCGTGGTGAGGGCGGTAAAGGGTCGAAGTTT AGGAGGAGGAAGGCGAGCGTTGGCGTATCGGAGGTTGCGGATTG
  • GDF10_57726 2662 GDF10 GTTATAGTCGTTCGGAGTAGCGTAGAGTCGAGTCGAGTTCGAGTCGGCG CGTTGTTTTGGCGGATTCGCGTCGCGAAAGTTTGTAGTTTATTGCG
  • GDF10_57728 2662 GDF10 TAGTTGGGGTTCGGGTTTCGGGTTGGTTCGAGCGGGGATATGAGTTATG GCGTGGTGAGGGCGGTAAAGGGTCGAAGTTTAG
  • GDF10_57734 2662 GDF10 TTTTTATATTCGCGGGCGTATATTTCGGCGCGCGTACGTTGTTATATACG GGCGTACGTATACGGTAGTCGGGTTAGGGACGATTTT
  • GJB2_22306 2706 GJ B2 TGCGGAGTATAGAGGATAACGATTATAGTTATTTTTGAATTTCGTTTACGG
  • GPNMB_52603 10457 GPNMB TTAATTTTTAGTTTTTCGATTGCGATTGCGATTCGGGAAAACGTTTACGTT
  • GPNMB_52605 10457 GPNMB GAAGCGGTTAAAGGCGTAGCGGTTTTTGGTTAGAGTCGTAGAGGTTTGA
  • GREM1_29777 26585 GREM1 GAATTTGGTACGATTTTACGGAGATTTCGTTTTTTTTAGCGTAGTTTTCGT
  • Gst-Pi New1 2950 GSTP1 TCGTTATTAGTGAGTACGCGCGGTTCGCGTTTTCGGGGATGGGGTTTAG AGTTTTTAGTATGGGGTTAATTCGTAGT ATTAGGTTCGGGTTTTCGGTAG GGTTTTTCGTTTATT 165 Gst-P ⁇ _New3 2950 GSTP1 ATTTAGTATTGGGGCGGAGCGGGGCGGGATTATTTTTATAAGGTTCGGA
  • HIST1 H3G_57040 8355 HIST1H3G CGATTTTTCGTATTAGGCGTTGGAAAGGTAATTTGCGGATTAGTAGTTTA
  • HIST1 H3G_57042 8355 HIST1H3G CGGGTGAAAGTAGGCGGTTTTTAAAAGAGTTTTTTTAAGTTGGATAGAAT
  • HMG20B_57034 10362 HMG20B GGTGAAAATAGTCGCGGAATTTCGGGTTTTGAGAGGGGGCGGGGGTGT
  • HOXA10_57000 3206 HOXA10 GTTGTTTTATTGCGTTTGTCGTTTAGCGTGGGGAAGAGTTCGTAGTTTTG TAGTTCGTAGGGTAGGTCGGCGGCGGGCGGTAGGTAGATTTCG
  • HOXA10_57002 3206 HOXA10 TTTATTCGGTAAGATCGGGGCGCGTTTAGTTATAGGTTTATGGGCGAGG GTTCGTAGTCGTGCGTTTCGGGATTTAGATT
  • HOXA5_1 3202 HOXA5 GTAGTTCGGGTTATTTGGAT AGCGATCGTAAAATGAGTTTATAAAAT AAG
  • HUS1B_12677 135458 HUS1B GTTGGGCGGTAGGTAGTTTCGTTATATTTTTTTGGGAAGTATTCGTACGG
  • ICAM1 3383 ICAM1 TAAAGACGTTTTCGCGGTTAAGGTCGAAAGGGGAAGCGAGGAGGTCGTC
  • GAATTAGAGAGT 205 ING1_24685 3621 ING1 TCGAAATAGAATTGGTAATCGTAGTAGTTAATTTATTTGTTAATATTATTTT

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Abstract

Several genes are identified as being differentially methylated in breast cancers. This information is useful for breast cancer screening, risk-assessment, prognosis, disease identification, disease staging, and identification of therapeutic targets. The identification of genes that are methylated in breast cancer allows accurate and effective early diagnostic assays, methylation profiling using multiple genes and identification of new targets for therapeutic intervention.

Description

MARKERS FOR IMPROVED DETECTION OF BREAST CANCER
[01] This invention was made using U.S. government funds under grant ESl 1858 from the National Institute of Environmental Health Sciences under grant CA043318 from the National Cancer Institute. The U.S. government retains certain rights to the invention under the terms of these grants.
FIELD OF THE INVENTION
[02] The present invention relates generally to cancer diagnosis and therapeutics. In particular, it relates to the identification of a cell proliferative disorder of breast by determining aberrant DNA methylation patterns of particular genes in breast cancer and pre-cancer.
BACKGROUND
[03] Breast cancer is one of the most significant health concerns. Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer death in women. One in eight women (13% of women) will develop breast cancer during her lifetime. Additionally, a small number of men are diagnosed with or die from breast cancer.
[04] Early breast cancer usually does not cause pain and may show no symptoms at all. The first signs come from self-examination or clinical breast examination, and regular screening mammography that often detect breast cancers before they cause any symptoms. Unfortunately mammography lacks both sensitivity and specificity, leading to a delayed diagnosis and risk of more advanced disease. The most widespread cause of the lower sensitivity is dense breast tissue which is common in younger women (-50%) and women with a strong family history of breast cancer; in these women the sensitivity can be as low as 35% -50%. Moreover, some fast-growing tumors may grow large or spread to other parts of the body before a mammogram detects them. Also, symptoms that are similar to those of breast cancer may be the result of non-cancerous conditions like infection or a cyst. Further tests, like histopathology of tissue removed during biopsy, are then needed to find out if abnormal cells are present. Efforts to develop improved methods for cancer detection have focused on cancer markers such as proteins that are uniquely expressed by cancerous cells, or differentially expressed by cancerous cells compared to normal cells. An overview on techniques available for breast cancer detection and diagnosis is provided at the website of the National Cancer Institute. Despite recent advances in diagnosis and treating breast cancer, the prevalence of the disease has been steadily rising. Thus, there continues to be a need for more sensitive and specific methods to early detect breast cancer and to complement mammography in high-risk women.
[05] It is widely accepted that loss of tumor suppressor function leads to the initiation and progression of human cancer. Inactivation of tumor suppressor genes can result from both genetic mechanisms such as mutation and epigenetic mechanisms such as DNA hypermethylation (Ponder B.A, 2001; Herman et al., 2003). Alternatively, up-regulation of expression of a number of genes (e.g. MAGE-Al) has been associated with initiation and progression of human cancer. Identification of these genes provides insight into the biological processes underlying oncogenesis and is useful for developing new therapeutic and diagnostic modalities.
[06] To document genetically altered genes in tumors, Sjδblom et al. (Sjδblom et al, 2006) sequenced 13,023 human genes in breast and colon cancer and identified 1,149 genes that harbored somatic mutations. Through statistical analysis, they showed that the majority of these changes were passenger mutations and that 189 genes were likely selected for during tumorigenesis (candidate cancer genes, CAN). For virtually all of the newly discovered mutations, the frequencies in each tumor type were low - in the range of 5 to 15%, and the vast majority of these mutations were heterozygous missense mutations. Thus, it is difficult to know whether each mutation conveys an oncogenic or tumor suppressor function. Most of the mutations identified in breast cancers were not present in colon tumors and vice versa, suggesting that the mutational spectrum is highly tumor-type specific. Similarly, Greenman et al. (Greenman et al., 2007) demonstrated that the mutational spectrum of protein kinases in tumors is highly variable and that mutations in a large number of cancer genes are operative in human tumors. Again, it is unknown whether most of the mutated genes are oncogenes or tumor suppressors.
[07] Epigenetic silencing is a second mechanism by which abnormal gene inactivation can occur in cancer. A predominant mode of epigenetic alteration in cancer is gene silencing via CpG island promoter hypermethylation (henceforth called hypermethylation). Hypermethylation acts by recruiting methyl-cytosine-binding proteins and histone deacetylases, which in a coordinated fashion, modify nucleosomes to form transcriptionally repressive chromatin (Busslinger et al., 1983; Nan et al., 1998). Repressive histone marks such as methylation of lysine-9 on histone 3 (H3K9) may initiate and help maintain this state of repression (Jenuwein et ah, 2006; Barski et ah, 2007). This results in the activation of many oncogenes and silencing of tumor suppressors to promote proliferation of abnormal cells. Alternatively, hypomethylation of a gene may lead to aberrant expression of certain antigens in a wide variety of tumors. These epigenetic abnormalities (hyper- and hypomethylation) could cooperate with genetic alterations to effect aberrant gene function that results in cancer.
[08] Schuebel et al. (Schuebel et al, 2007) developed a transcriptome-wide approach to identify genes affected by promoter CpG island hypermethylation and transcriptional silencing in colorectal cancer. They demonstrated when directly compared to gene mutations, much larger number of genes hypermethylated in individual tumors, and much higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. They concluded that genome wide strategies for mapping aberrant gene changes in cancer should take into account that mutated genes should also be examined for promoter DNA hypermethylation.
[09] DNA methylation markers have been evaluated as potential genetic markers for detection of cancer because they offer certain advantages when compared to mutation markers: DNA hypermethylation appears to be an early event in the etiology of carcinogenesis and appears to precede apparent malignancy in many cases (Esteller et ah, 2001). The methylation profile is in many cases tissue- and tumor-type specific and is also preserved in purified isolated DNA. In addition, methylation markers may serve for predictive purposes as they often reflect the sensitivity to therapy or duration of patient survival. DNA hypermethylation is also prevalent in the highly aggressive HER2/Neu-positive breast cancers.
[10] An early diagnosis is critical for the successful treatment of many types of cancer. If the exact methylation profiles of breast tumors are available and drugs targeting the specific genes are obtainable, then the treatment of breast cancer could be more focused and rational. More sensitive methylation-based methods could help to identify those patients with a higher probability of cancer recurrence. Such products could help the physician prescribe the potentially most suitable drug treatment to the right patient earlier in the disease process. Therefore, the detection and mapping of novel methylation markers, is an essential step towards improvement of breast cancer prevention, screening and treatment. SUMMARY OF THE INVENTION
[11] The present invention is based on the finding that several genes are newly identified as being differentially methylated in breast cancers. This information is useful for breast cancer screening, risk-assessment, prognosis, disease identification, disease staging and identification of therapeutic targets. The identification of new genes that are methylated in breast cancer allows accurate and effective early diagnostic assays, methylation profiling using multiple genes and identification of new targets for therapeutic intervention.
[12] In one aspect, the invention provides for a method for identifying breast cancer or its precursor, or predisposition to breast cancer. Epigenetic modification is detected in a test sample containing breast cells or nucleic acids from breast cells. The epigenetic modification is of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. The epigenetic modification may be detected by determining the state of methylation of one or more nucleic acids from a subject. A difference in the state of methylation of one or more nucleic acids in a test sample of the subject compared to the state of methylation of one or more nucleic acids from a subject not having a cellular proliferative disorder of breast tissue is indicative of the test sample containing breast cells that are neoplastic, precursors to neoplastic, or predisposed to neoplasia, or as containing nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
[13] Methylation changes that develop in the process of carcinogenesis are not only ideal for screening purposes, but also interesting targets for monitoring staging. Accordingly, the invention also provides for a method for determining the histopathological stage of breast cancer, comprising detecting epigenetic modification of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF 195; ZNF365; ZNF432; and ZNF655; wherein detection of the epigenetic change is indicative of the histopathological stage of the breast cancer.
[14] Epigenetic loss of gene function can be rescued by the use of DNA demethylating agents and/or DNA methyltransferase inhibitors and/or histone deacetylase (HDAC) inhibitor.
[15] According to another aspect of the invention a method is provided of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer. An epigenetically silenced gene is determined in a cell. The gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. Expression of a polypeptide encoded by the epigenetic silenced gene in the cell is restored by contacting the cell with one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor. Unregulated growth of the cell is thereby reduced or inhibited. Alternatively, reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer is accomplished by introduction of a polynucleotide encoding a polypeptide into the cell. The polypeptide is encoded by said gene. The polypeptide is expressed in the cell thereby restoring expression of the polypeptide in the cell.
[16] Yet another aspect of the invention is a method of treating a cancer patient. A cancer cell in the patient is determined to have an epigenetic silenced gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT 14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. One or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor is administered to the patient in sufficient amounts to restore expression of the epigenetic silenced gene in the patient's cancer cells.
[17] Yet another aspect of the invention is a method of treating a cancer patient. A cancer cell in the patient is determined to have an epigenetic silenced gene selected from those shown in HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. A polynucleotide encoding a polypeptide is administered to the patient. The polypeptide is encoded by the epigenetic silenced gene. The polypeptide is expressed in the patient's tumor thereby restoring expression of the polypeptide in the cancer.
[18] In still another aspect of the invention a method is provided for selecting a therapeutic strategy for treating a cancer patient. A gene is identified whose expression in cancer cells of the patient is reactivated by a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, or a histone deacetylase (HDAC) inhibitor. The gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. A therapeutic agent that increases expression of the gene for treating said cancer patient is selected for the cancer patient.
[19] Another embodiment of the invention is a kit for assessing methylation in a test sample. The kit comprises at least the following reagents: a reagent that (a) modifies methylated cytosine residues but not non-methylated cytosine residues, or that (b) modifies non- methylated cytosine residues but not methylated cytosine residues; and a pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp of said gene's transcription start site, said gene being selected from those shown in HOXDl; SLC2A14; NEFH; HOXA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; HOXA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; COX7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; HOXA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655.
[20] According to a further aspect, the invention provides oligonucleotide primers and/or probes and their sequences for use in the methods and assays of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[21] FIG. 1A-1B Strategy used to identify common targets of mutation and promoter hypermethylation.
[22] FIG. IA, Large-scale sequencing of breast and colon cancers identified 189 genes cancer genes (CAN genes) as reported by Sjδblom et al. Candidate hypermethylated genes were identified via expression microarray analysis. Genes without promoter CpG islands were excluded resulting in 36 target genes. [23] FIG. IB, Frequency of promoter methylation in the 36 CAN genes that are subject to hypermethylation. X-axis denotes percent methylation. Methylation status was determined in the 6 colorectal cancer cell lines and 11 breast cancer cell lines as indicated in the example section.
[24] FIG. 2 : Primers used for Methylation-specific PCR of the 36 target genes. (SEQ ID NO: 1305-1448, respectively)
[25] FIG. 3. : Promoter methylation frequency in primary colon and breast tumors: 18 out of the 36 common target genes methylated in either breast cancer cell lines, colon cancer cell lines or both, showed cancer-specific methylation. Y-axis denotes percent methylation.
[26] FIG. 4 : Mutation and methylation plot of the 18 genes showing cancer-specific methylation..
[27] FIG. 5 : Primary breast tumor grade or stage correlation. Y-axis denotes percent methylation for the different tumor stages/grades.
[28] FIG. 6A-6B: FIG. 6A. Position of the different primers relative to the TSS.
Multiple primer designs are displayed by blue boxes and red boxes (=final primer pairs retained for the assays). The exon of PPP1R13B is indicated in green. The number of CpG count is spotted in blue over a region of 20Kb. FIG. 6B. Sequences of the different primer sets, sense (SEQ ID NO: 1273-1280, respectively) and antisense (SEQ ID NO: 1281-1288, respectively) and amplicons. (SEQ ID NO: 1289-1296, respectively)
[29] Fig. 7: Methylation frequency SLC2A14, NEFH and TF. Percentage methylation (Y-axis) is shown for each marker (X-axis)
DETAILED DESCRIPTION OF THE INVENTION
[30] We describe a whole human transcriptome microarray screen to identify genes silenced by promoter hypermethylation in human breast cancer. The microarray screen identifies hypermethylated genes that are re-expressed following treatment with the DNA methyltransferase (DNMT) inhibitor 5-deoxyazacytidine (DAC) but not following treatment with the HDAC I/II inhibitor trichostatin A (TSA) alone. Based on the sensitivity differences observed between DKO (DNMT double knockout) and DAC induced gene increases, we designated a list— top tier and next tier— of candidate cancer genes in tumors with a high efficiency of validation.
[31] By comparing the list of candidate hypermethylated genes with mutated genes recently identified in breast cancer (3), we define, for any human cancer type for which representative cell culture lines are available, a substantial fraction of the cancer gene promoter CpG island DNA hypermethylome. We document that many more epigenetically versus genetically altered genes exist in any given tumor. The importance of this fact emerges in our finding that for newly discovered genes that are affected by both mechanisms, the incidence for hypermethylation of any given gene among breast cancers appears to be much higher than for mutations. Therefore, within a given cancer type, one may markedly underestimate both the full range of gene alterations and associated abnormalities of cellular pathways by failing to screen for both genetic and epigenetic abnormalities. The data also indicate that assessing both mechanisms for loss of gene function indicates far more sharing among individual breast tumors for pathway disruption than genetic analyses alone would predict. Our findings emphasize that optimal approaches to grouping of tumors according to molecular alterations in key pathways should depend on defining both genetic and epigenetic gene changes. Thus, our findings should encourage any genome wide strategies for mapping aberrant gene changes in cancer to take into account that mutated genes should be examined for promoter DNA hypermethylation and DNA hypermethylated genes should be put in a priority position for sequencing to find mutations.
[32] A further substantial fraction of the cancer gene promoter CpG island DNA hypermethylome is defined through comparison of the hypermethylome genes found by the micro human transcriptome microarray screen and their specificity for certain cell lines such as metastatic cell lines but not for non-metastatic cell lines. Resulting methylation markers are useful in identifying cancers with high metastatic potential and may provide guidance to a preferred method of treatment. For instance, for cancers with high metastatic potential it may be indicated to have radiotherapy treatment following the resection of the cancer.
[33] We also used a Genome-wide Promoter Alignment approach with the capacity to define still a further substantial fraction of the cancer gene promoter CpG island DNA hypermethylome. Markers clustering with known methylation markers might indicate towards common mechanisms underlying the hypermethylation event and thus identify novel genes that are more methylation-prone.
[34] Studies of the genes defined by the different approaches will contribute to understanding the molecular pathways driving tumorigenesis, provide useful new DNA hypermethylation biomarkers to monitor cancer risk assessment, early diagnosis, and prognosis, and permit better monitoring of gene re-expression during cancer prevention and/or therapy strategies
[35] Using the aforementioned techniques, we have discovered a set of genes whose transcription is epigenetically silenced in cancers, cancer precursors, and pre-cancers. The genes include: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT 14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. Detection of epigenetic silencing of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of such genes can be used as an indication of cancer or pre-cancer or risk of developing cancer. The accession numbers corresponding to the listed genes can be found at the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health website.
[36] The term "silenced" or "silencing", when used in reference to a gene, means that the gene is not being transcribed, or is being transcribed at a reduced level when compared to the transcription level of the same gene in a corresponding control cell, e.g., a healthy cell, a non-cancerous cell, a non-infected cell. A consequence of the silencing of a gene is that a cell comprising the silenced gene has reduced levels of, or completely lacks, a polypeptide encoded by the gene, i.e. the gene product, such that any function normally attributed to the gene product in the cell is reduced of absent.
[37] Assays that recognize only a single marker of the listed markers may be of limited predictive value. A multiplexed analytical approach is particularly suitable for cancer diagnostics, and thus a panel approach may be more consistent with the heterogeneous nature of cancer. Combinations of markers may be applied as desired. Multiplexing in connection to epigenetic silencing of genes has been described in WO05042713 and such methods are applicable in the present invention.
[38] The cancer to be identified is preferably a breast cancer. There are two major groups of breast cancer: noninvasive carcinoma and invasive carcinoma. The noninvasive carcinomas include lobular carcinoma in situ and ductal carcinoma in situ. Unfortunately, breast cancers often grow through the basement membrane and roughly 95% of all breast cancers are infiltrating or invasive carcinomas. The most common type of invasive breast cancer (about 75%) is invasive ductal carcinoma, arising in the milk ducts and spreading through the duct walls. Invasive lobular carcinoma originates in the milk glands and accounts for 10 to 15% of invasive breast cancers. Less common types of invasive breast cancer include the following: inflammatory breast cancer, Paget's disease of the nipple, medullary carcinoma, mucinous carcinoma, phyllodes tumor, and tubular carcinoma. Rarely (about 1%), sarcomas (cancer of the connective tissue) develop in the breasts. Individuals may develop one, the other, or a combination of invasive and noninvasive breast cancer.
[39] Epigenetic silencing of a gene can be determined by any method known in the art. One method is to determine that a gene which is expressed in normal cells or other control cells is less expressed or not expressed in tumor cells. This method does not, on its own, however, indicate that the silencing is epigenetic, as the mechanism of the silencing could be genetic, for example, by somatic mutation. One method to determine that the silencing is epigenetic is to treat with a reagent, such as DAC (5'-deazacytidine), or with a reagent which changes the histone acetylation status of cellular DNA or any other treatment affecting epigenetic mechanisms present in cells, and observe that the silencing is reversed, i.e., that the expression of the gene is reactivated or restored.
[40] Another means to detect epigenetic silencing is to determine the presence of methylated CpG dinucleotide motifs in the silenced gene. Methylation of a CpG island at a promoter usually prevents expression of the gene. The islands can surround the 5' region of the coding region of the gene as well as the 3' region of the coding region. Thus, CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns. All of these regions can be assessed to determine their methylation status. Typically these reside near the transcription start site and surround the TTS, for example, within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp.
[41] Once a gene has been identified as the target of epigenetic silencing in tumor cells, determination of reduced expression can be used as an indicator of epigenetic silencing. Expression of a gene can be assessed using any means known in the art. Typically expression is assessed and compared in test samples and control samples which may be normal, non-malignant cells. Either mRNA or protein can be measured. Methods employing hybridization to nucleic acid probes can be employed for measuring specific mRNAs. Such methods include using nucleic acid probe arrays (microarray technology), in situ hybridization, and using Northern blots. Messenger RNA can also be assessed using amplification techniques, such as RT-PCR. Sequencing-based methods are an alternative; these methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS). Differential display techniques provide another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest. Specific proteins can be assessed using any convenient method including immunoassays and immuno- cytochemistry but are not limited to that. Most such methods will employ antibodies which are specific for the particular protein or protein fragments. The sequences of the mRNA (cDNA) and proteins of the markers of the present invention are known in the art and publicly available.
[42] The term "microarray" refers broadly to both "DNA microrarray" and "DNA chips", and encompasses all art-recognized solid supports and methods for affixing nucleic acid molecules thereto or synthesis of nucleic acids thereon.
[43] Methylation-sensitive restriction endonucleases can be used to detect methylated CpG dinucleotide motifs. Such endonucleases may either preferentially cleave methylated recognition sites relative to non-methylated recognition sites or preferentially cleave non- methylated relative to methylated recognition sites. Non limiting examples of the former are Aat II, Ace III, Ad I, AcI I, Age I, AIu I, Asc I, Ase 1, AsiS I, Ban I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrV I, BssK 1, BstB I, BstN I, Bs I, CIa I, Eae I, Eag I, Fau I, Fse I, Hha I, mPl I, HinC II, Hpa 11, Npy99 I, HpyCAIV, Kas I, Mbo I, MIu I, MapA 1 1. Msp I, Nae I, Nar I, Not 1, PmI I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, SfI I, Sfo I, SgrA I, Sma I SnaB I, Tsc I, Xma I, and Zra I. Non limiting examples of the latter are Ace II, Ava I, BssH II, BstU I, Hpa II, Not I and Mho I.
[44] Alternatively, chemical reagents can be used which selectively modify either the methylated or non-methylated form of CpG dinucleotide motifs. Modified products can be detected directly, or after a further reaction which creates products which are easily distinguishable. Means which detect altered size and/or charge can be used to detect modified products, including but not limited to electrophoresis, chromatography, and mass spectrometry. Examples of such chemical reagents for selective modification include hydrazine and bisulfite ions. Hydrazine-modified DNA can be treated with piperidine to cleave it. Bisulfite ion-treated DNA can be treated with alkali. Other means for detection which are reliant on specific sequences can be used, including but not limited to hybridization, amplification, sequencing, and ligase chain reaction. Combinations of such techniques can be used as is desired.
[45] The principle behind electrophoresis is the separation of nucleic acids via their size and charge. Many assays exist for detecting methylation and most rely on determining the presence or absence of a specific nucleic acid product. Gel electrophoresis is commonly used in a laboratory for this purpose.
[46] One may use MALDI mass spectrometry in combination with a methylation detection assay to observe the size of a nucleic acid product. The principle behind mass spectrometry is the ionizing of nucleic acids and separating them according to their mass to charge ratio. Similar to electrophoresis, one can use mass spectrometry to detect a specific nucleic acid that was created in an experiment to determine methylation. See Tost, J. et al. Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry.
[47] One form of chromatography, high performance liquid chromatography, is used to separate components of a mixture based on a variety of chemical interactions between a substance being analyzed and a chromatography column. DNA is first treated with sodium bisulfite, which converts an unmethylated cytosine to uracil, while methylated cytosine residues remain unaffected. One may amplify the region containing potential methylation sites via PCR and separate the products via denaturing high performance liquid chromatography (DHPLC). DHPLC has the resolution capabilities to distinguish between methylated (containing cytosine) and unmethylated (containing uracil) DNA sequences. Deng, D. et al. describes simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography.
[48] Hybridization is a technique for detecting specific nucleic acid sequences that is based on the annealing of two complementary nucleic acid strands to form a double-stranded molecule. One example of the use of hybridization is a microarray assay to determine the methylation status of DNA. After sodium bisulfite treatment of DNA, which converts an unmethylated cytosine to uracil while methylated cytosine residues remain unaffected, oligonucleotides complementary to potential methylation sites can hybridize to the bisulfite-treated DNA. The oligonucleotides are designed to be complimentary to either sequence containing uracil or sequence containing cytosine at the original CpG islands, representing unmethylated and methylated DNA, respectively. Computer-based microarray technology can determine which oligonucleotides hybridize with the DNA sequence and one can deduce the methylation status of the DNA. [49] An additional method of determining the results after sodium bisulfite treatment would be to sequence the DNA to directly observe any bisulfite-modifications. Pyrosequencing technology is a method of sequencing-by-synthesis in real time. It is based on an indirect bioluminometric assay of the pyrophosphate (PPi) that is released from each deoxynucleotide (dNTP) upon DNA-chain elongation. This method presents a DNA template-primer complex with a dNTP in the presence of an exonuclease-deficient Klenow DNA polymerase. The four nucleotides are sequentially added to the reaction mix in a predetermined order. If the nucleotide is complementary to the template base and thus incorporated, PPi is released. The PPi and other reagents are used as a substrate in a luciferase reaction producing visible light that is detected by either a luminometer or a charge-coupled device. The light produced is proportional to the number of nucleotides added to the DNA primer and results in a peak indicating the number and type of nucleotide present in the form of a pyrogram. Pyrosequencing can exploit the sequence differences that arise following sodium bisulfite-conversion of DNA.
[50] In a particular aspect, present methods use amplification techniques in a reaction for creating distinguishable products. Multiple amplification techniques are known. Some of these techniques employ PCR. Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), transcription amplification (Kwoh et al. 1989; WO88/10315), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO90/06995), nucleic acid based sequence amplification (NASBA) (US Patent Nos 5,409,818; 5,554,517; 6,063,603), nick displacement amplification (WO2004/067726).
[51] Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to PCR primer design. In the first approach, the primers do not themselves "cover" or hybridize to any potential sites of DNA methylation; sequence variation at sites of differential methylation are located between the two primers. Such primers are used in bisulphite genomic sequencing, COBRA, Ms-SNuPE. In the second approach, the primers are designed to anneal specifically with either the methylated or unmethylated version of the converted sequence. If there is a sufficient region of complementarity, e.g., 12, 15, 18, 20 or more nucleotides, to the target, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats. The oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
[52] One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After primer hybridization, an amplification reaction can be performed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not. For example, bisulfite ions convert non- methylated cytosine bases to uracil bases. Uracil bases hybridize to adenine bases under hybridization conditions. Thus an oligonucleotide primer which comprises adenine bases in place of guanine bases would hybridize to the bisulfite-modified DNA, whereas an oligonucleotide primer containing the guanine bases would hybridize to the non-converted (initial methylated) cytosine residues in the modified DNA. Amplification using a DNA polymerase and a second primer yield amplification products which can be readily observed. This method is known as MSP (Methylation Specific PCR; Patent Nos 5,786,146; 6,017,704; 6,200,756). Preferred primers and primer sets for assessing the methylation status of the concerned gene by way of MSP are provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288). The amplification products can be optionally hybridized to specific oligonucleotide probes which may also be specific for certain products. Alternatively, oligonucleotide probes can be used which will hybridize to amplification products from both modified and non- modified DNA.
[53] Thus, present invention provides for a method for detecting breast cancer or its precursor, or predisposition to breast cancer in a test sample containing breast cells or nucleic acids from breast cells comprising:
contacting a methylated CpG-containing nucleic acid of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5;
ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl;
C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl;
CNIH3; CNNl; CNN3; COL7A1; COL9A3; COX7A1; CRIPl; CSPG2; CST6;
CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27;
DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2;
EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4;
FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7;
GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO;
HOXA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7;
ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6;
LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846;
MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL;
NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA;
PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B;
PPPl Rl 4A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1;
RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B;
SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9;
SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2;
TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl;
WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655 with bisulfite to convert unmethylated cytosine to uracil; detecting the methylated CpGs in the nucleic acid by contacting the converted nucleic acid with oligonucleotide primers whose sequence discriminates between the bisulfite-treated methylated and unmethylated version of the converted nucleic acid; and identifying the test sample as containing cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia, or as containing nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
[54] Modified and non-modified DNA can be distinguished with use of oligonucleotide probes which may also be specific for certain products. Such probes can be hybridized directly to modified DNA or to amplification products of modified DNA. Probes for assessing the methylation status of the concerned gene will specifically hybridize to the converted sequence but not to the corresponding non converted sequence. Probes are designed to anneal specifically with the converted sequence representing either the methylated or unmethylated version of the DNA. Preferred converted sequences are provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495- 1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288). Preferred probes anneal specifically with the converted sequence representing the methylated version of the DNA, in particular those sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501- 1503), and Figure 6B (SEQ ID NO: 1289-1304). Oligonucleotide probes can be labeled using detection systems known in the art. These include but are not limited to fluorescent moieties, radioisotope labeled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.
[55] Real time chemistry allows for the detection of PCR amplification during the early phases of the reactions, and makes quantitation of DNA and RNA easier and more precise. A few variants of real-time PCR are well known. They include Taqman® (Roche Molecular Systems), Molecular Beacons®, Amplifluor® (Chemicon International) and Scorpion® DzyNA®, Plexor™ (Promega) etc. The TaqMan® system and Molecular Beacon® system have separate probes labeled with a fluorophore and a fuorescence quencher. In the Scorpion® system the labeled probe in the form of a hairpin structure is linked to the primer.
[56] Quantitation in real time format may be on an absolute basis, or it may be relative to a methylated DNA standard or relative to an unmethylated DNA standard. The absolute copy number of the methylated marker gene can be determined; or the methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene with a known methylation (e.g. β-actin), or by using the ratio between the methylated marker and the sum of the methylated and the non- methylated marker.
[57] Real-Time PCR detects the accumulation of amplicon during the reaction, but alternatively end-point PCR fluorescence detection techniques may be used. Confirming the presence of target DNA at the end point stage may indeed be sufficient and it can use the same approaches as widely used for real time PCR. [58] DNA methylation analysis has been performed successfully with a number of techniques which are also applicable in present methods of the invention. These include the MALDI- TOFF, MassARRAY (Ehrich, M. et al 2005), MethyLight (Trinh B. et al 2001), Quantitative Analysis of Methylated Alleles (Zeschnigk M. et al.), Enzymatic Regional Methylation Assay (GaIm et al, 2002), HeavyMethyl (Cottrell, SE et al, 2004), QBSUPT, MS-SNuPE (Gonzalgo and Jones, 1997), MethylQuant (Thomassin H. et al. 2004), Quantitative PCR sequencing, and Oligonucleotide-based microarray systems (Gitan RS et al. ,2006)
[59] Alternatively, identification of methylated CpG dinucleotides may utilize the ability of the MBD domain of the McCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999). Restriction enconuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Variants of this method have been described and may be used in present methods of the invention.
[60] Test samples for diagnostic, prognostic, or personalized medicine uses can be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin embedded colon, rectum, small intestinal, gastric, esophageal, bone marrow, breast, ovary, prostate, kidney, lung, brain or other organ tissues, from lymph nodes, or from a body fluid such as blood, serum, lymph, cerebrospinal fluid, saliva, sputum, bronchial -lavage fluid, ductal fluids stool, urine, or semen. A test sample obtainable from such specimens or fluids includes detached tumor cells or free nucleic acids that are released from dead or damaged tumor cells. Nucleic acids include RNA, genomic DNA, mitochondrial DNA, single or double stranded, and protein-associated nucleic acids. Any nucleic acid specimen in purified or non-purified form obtained from such specimen cell can be utilized as the starting nucleic acid or acids. Particular suitable for breast cancer assays are blood derived samples such as serum and plasma. Alternatively, fluid within the mammary glands is used. Such fluid can be obtained from the breast e.g. by nipple aspiration of the milk ducts or by ductal lavage of at least one breast milk duct or from spontaneous nipple discharge. Administration of oxytocin may be used to stimulate expression of mammary fluid from a nipple of the patient. Collection of the mammary fluid may require the use of a fluid collector such as a breast pump. [61] Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA, and leads to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions. Analysis of RARbeta2 and RASSFlA methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women (Skvortsova et α/.32006). Thus, in order to provide sensitive and accurate detection and discrimination of malignant and benign breast tumours, it may be beneficial to conduct methylation-specific PCR of the listed genes on the total cirDNA, eventually combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood.
[62] In one particular aspect, primers and probes useful in MSP carried out on the gene selected from HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EY A4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655 are provided. The primers specifically hybridize to the sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304). Preferred primers comprise, consist essentially of, or consist of the sequences provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288). The probes specifically hybridize to the sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304). Preferred probes (5' - 3') may comprise, consist essentially of or consist of the sequences represented by SEQ ID NO. 1485-1494. Alternative fluorescent donor and acceptor moieties/FRET pairs may be utilized as appropriate. In addition to being labeled with the fluorescent donor and acceptor moieties, the primers and probes may include modified oligonucleotides and other appending groups and labels provided that the functionality as a primer and/or probe in the methods of the invention is not compromised.
[63] When very low concentrations must be detected, an enrichment step prior to the amplification step may be appropriate. The nested MSP approach (US 7,214,485) for instance may provide a solution. Alternatively, the generation and amplification of a DNA library before testing for methylation of any specific gene may be required. Suitable methods on whole genome amplification and libraries generation for such amplification (e.g., Methylplex and Enzyplex technology, Rubicon Genomics) are described in US2003/0143599, WO2004/081225 and WO2004/081183. In addition, WO2005/090507 regards library generation/amplification methods that require either bisulphite conversion or non-bisulphite based application. Bisulphite treatment may occur before or after library construction and may require the use of adaptors resistant to bisulphite conversion. Meth- DOP-PCR (Di Vinci et al, 2006), a modified degenerate oligonucleotide-primed PCR amplification (DOP-PCR) that is combined with MSP, provides another suitable method for specific detection of methylation in small amount of DNA. Improved management of patient care may require these existing methods and techniques to supplement the methods of the invention.
[64] To attain high rates of tumor detection, it may be necessary to combine the methods of the invention with established methods and/or markers for breast cancer identification. Methods of present invention are preferentially used in conjunction with one or more of the following methods and/or tumor markers:
- CA 15-3, CA 27.29, CEA, carcinoembryonic antigen (CEA), estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-I).
- Multiparameter assays for gene expression such as Oncotype DX assay (Genomic Health,Inc, Redwood city, CA, USA), Mammaprint assay (Agendia BV, Amsterdam).
- BRACAnafysis® assesses a woman's risk of developing breast or ovarian cancer based on detection of mutations in the BRCAl and BRCA2 genes.
- Approximately 25-30 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression of this receptor is associated with highly aggressive breast cancer that requires special treatment, increased disease recurrence and worse prognosis. DNA hypermethylation is prevalent in these highly aggressive HER2/Neu-positive breast cancers. To aid prognostication, it may be necessary to combine the methods of the invention with this established marker for breast cancer prognosis.
[65] Marker and assay details are available in the Guidelines Updated for Use of Tumor Markers in Breast Cancer at a website called MedScape™.
[66] Following diagnosis, treatment is often decided according to the stage of a cancer. The "stage" of a cancer is a descriptor (usually numbers I to IV) of how much the cancer has spread. The stage takes into account the size of a tumor, how deep it has penetrated, whether it has invaded adjacent organs, if and how many lymph nodes it has metastasized to, and whether it has spread to distant organs. Staging of cancer is important because the stage at diagnosis is the biggest predictor of survival, and treatments are often changed based on the stage. The "grade" of a cancer refers to cell appearance (differentiation) and DNA make up.
[67] Related hereto, the invention also provides for a method for determining the stage of cancer comprising determining epigenetic silencing of a gene in cancers, cancer precursors, and pre-cancers. The genes include: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. As shown in the examples, SYNEl and COL7A1 are preferentially methylated in advanced tumors. PTPRD, SYNEl, and EVL are preferentially hypermethylated in high grade tumors. These markers are preferred markers for determining the stage and/or grade of the breast cancer. Since grade is a strong predictor of local recurrence and metastasis, such markers are useful in predicting aggressive clinical behavior of the cancer and prognosis.
[68] Testing can be performed diagnostically or in conjunction with a therapeutic regimen. Testing can be used to monitor efficacy of a therapeutic regimen, whether a chemotherapeutic agent or a biological agent, such as a polynucleotide. Testing can also be used to determine what therapeutic or preventive regimen to employ on a patient. Moreover, testing can be used to stratify patients into groups for testing agents and determining their efficacy on various groups of patients. [69] According to another aspect of the invention a method is provided of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer. An epigentically silenced gene is determined in a cell. The gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. Expression of a polypeptide encoded by the epigenetic silenced gene in the cell is restored by contacting the cell with one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor. Unregulated growth of the cell is thereby reduced or inhibited.
[70] Another aspect of the invention is a method of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer. An epigenetic silenced gene is determined in the cell. The gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. A polynucleotide encoding a polypeptide is introduced into the cell. The polypeptide is encoded by said gene. The polypeptide is expressed in the cell thereby restoring expression of the polypeptide in the cell.
[71] Yet another aspect of the invention is a method of treating a cancer patient. A cancer cell in the patient is determined to have an epigenetic silenced gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; COX7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; HOXA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT 14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. One or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor is administered to the patient in sufficient amounts to restore expression of the epigenetic silenced gene in the patient's cancer cells.
[72] Yet another aspect of the invention is a method of treating a cancer patient. A cancer cell in the patient is determined to have an epigenetic silenced gene selected from those shown in HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. A polynucleotide encoding a polypeptide is administered to the patient. The polypeptide is encoded by the epigenetic silenced gene. The polypeptide is expressed in the patient's tumor thereby restoring expression of the polypeptide in the cancer.
[73] In still another aspect of the invention a method is provided for selecting a therapeutic strategy for treating a cancer patient. A gene is identified whose expression in cancer cells of the patient is reactivated by a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, or a histone deacetylase (HDAC) inhibitor. The gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT 14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655. Thus, a therapeutic agent which increases expression of the gene for treating said cancer patient is selected for the cancer patient.
[74] Demethylating agents can be contacted with cells in vitro or in vivo for the purpose of restoring normal gene expression to the cell. Suitable demethylating agents include, but are not limited to 5-aza-2'-deoxycytidine, 5-aza-cytidine, Zebularine, procaine, and L- ethionine. This reaction may be used for diagnosis, for determining predisposition, and for determining suitable therapeutic regimes. If the demethylating agent is used for treating breast cancer, expression or methylation can be tested of a gene selected from HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655.
[75] Another embodiment of the invention is a kit for assessing methylation in a test sample. Kits according to the present invention are assemblages of reagents for testing methylation. They are typically in a package which contains all elements, optionally including instructions. The package may be divided so that components are not mixed until desired. Components may be in different physical states. For example, some components may be lyophilized and some in aqueous solution. Some may be frozen. Individual components may be separately packaged within the kit. The kit may contain reagents, as described above for differentially modifying methylated and non-methylated cytosine residues. Desirably the kit will contain oligonucleotide primers which specifically hybridize to regions within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp kb of the transcription start sites of the genes/markers: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; COX7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; SOCS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655.
[76] Typically the kit will contain both a forward and a reverse primer for a single gene or marker. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats. The oligonucleotide primers may or may not be such that they are specific for modified methylated residues. The kit may optionally contain oligonucleotide probes. The probes may be specific for sequences containing modified methylated residues or for sequences containing non-methylated residues. The kit may optionally contain reagents for modifying methylated cytosine residues. The kit may also contain components for performing amplification, such as a DNA polymerase and deoxyribonucleotides. Means of detection may also be provided in the kit, including detectable labels on primers or probes. Kits may also contain reagents for detecting gene expression for one of the markers of the present invention. Such reagents may include probes, primers, or antibodies, for example. In the case of enzymes or ligands, substrates or binding partners may be sued to assess the presence of the marker.
[77] According to a further aspect, the invention provides for oligonucleotide primers and/or probes and their sequences for use in the methods and assays of the invention. Preferred primers and their sequences bind to at least one of the polynucleotide sequences listed in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304) or to the complement sequence thereof. Most preferred primers and their corresponding sequences are listed in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495- 1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288). Preferred probes and their sequences bind to at least one of the polynucleotide sequences listed in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304) or to the complement sequence thereof. Preferred probes (5' - 3') may comprise, consist essentially of or consist of the sequences represented by SEQ ID NO. 1485-1494, or the complement sequence thereof. Related to this, the invention also provides for an isolated polynucleotide which consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-1503.
[78] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
Table IA. MSP assay and primer details:
Official
Gene Sense Primer Sequence (5' - 3') Antisense Primer Sequence (5' -
Mr. Assay Name Gene ID SEQ ID NO'S 1 - 424 3') SEQ ID NO'S 425 - 848 Symbol
1 ABHD3_11682 171586 ABHD3 GATATTCGTCGTAGAGCGGAA CAAAAATAAACCGAAAACTAACGA
2 ABHD3_55542 171586 ABHD3 GGTTTTGCGTTATTTTCGGT CTTATCCTAAACTTCAACGTCGC
3 ACP5_55536 54 ACP5 TTTTGTATTTGCGGTCGGT AAAAACTCCTAAATATCCGCGA
4 ACP5_55540 54 ACP5 CGGTTTATTTTATAGATGCGGAG AAAAACGAACGCCTCGACC
5 ACTN2_55534 88 ACTN2 GCGGCGCGGTTATTAAGTC ACGACACGCCTAAACGCTA
6 ACTN2_55535 88 ACTN2 GTGTTTTGTTTGTTTTCGGAC TAACCGCGTAACCTAACGACT
7 ADAM23_2 8745 ADAM23 GAAGGACGAGAAGTAGGCG CTAACGAACTACAACCTTACCGA
8 ADAM23_A 8745 ADAM23 GAGGTTTTAAGTTGGCGGAGC ACTCGAAACTAAACGACGCCC
9 ADAMTS15_56144 170689 ADAMTS15 TAGTAGAAGTATGGCGTCGGG TCCAAAATACGAACTACACGAAA
10 ADAMTS15_56145 170689 ADAMTS15 GAGTTTTTGATTTTCGGAGTTTTC AATAATCGTTCCCATCCGACT
11 ADAMTS18_56138 170692 ADAMTS18 CGTTTTAGTTTCGTTAGGTTTTTC ATAAAATACGCCCTCCTACTCG
12 ADAMTS18_56141 170692 ADAMTS18 GGTGTCGTAAATTGTAGTTCGG CGTTCACATCGCAACGAAA
13 ADRA2A_57683 150 ADRA2A GGTAGAGTTCGCGTTTTAGTTTC TCGAAAATCCTATATCTATCGCC
14 ADRA2A_57684 150 ADRA2A GTTTTTGTTTTAATTCGCGTTG ATTCTCCTAAACGTCCGCCT
TTATATGTCGGTTACGTGCGTTTAT
15 APC(2) 324 APC GAACCAAAACGCTCCCCAT
AT
16 APC2 10297 APC2 GTCGTTTGTTTAGGTTCGGATC GACCCGAAATAACCTCGAAACG
17 APC2_56103 10297 APC2 TCGGATGGTGAAGTTCGTG AAACTAAATCCCGATAAACTAAACG
18 AQP5_56090 362 AQP5 TCGGGATCGAGTTTCGTTT CCCTACGCATAATACGCCC
19 AQP5_56093 362 AQP5 AAGAAAGTTCGGAGTAGCGAGA ACCCTAAAACCCGTAAACGAC
20 ARMCX2_13030 9823 ARMCX2 TTTTACGTGATTAGGAGTTGACG ATTAAACGCACGACAACGAA
21 ARMCX2_13032 9823 ARMCX2 TGGCGATGTAGTTTTTACGTG ATTAAACGCACGACAACGAA
22 ATP2A2_23347 488 ATP2A2 TTTTTAATTCGTTTTGCGGAG CCTCCTCTTACGACCGCTT
23 ATP2A2_23355 488 ATP2A2 GAGGGTTCGAGAAGCGAAG CCGACAACTACGAACGAAA
24 ATXN1_56027 6310 ATXN 1 TACGGGGATAGATTTGGGAGC ATAAACCGACAAAAACCTCGATA
25 ATXN1_56031 6310 ATXN 1 AATTAAGAGCGGAGTAACGAA ACCTTCTCCTCGAAAAACCGA
26 ATXN1_56032 6310 ATXN 1 GGAGTAGTTGGTTGTCGTCGT CCGTACTCTATACGACTCGCC
27 AXIN2_56020 8313 AXI N2 GAGAGATAGAGAGATTACGTCGATT GAAAAACAAATCACCAACATAACG
28 BACH1_56015 571 BACH1 TTTTGTAATTTTTCGCGTGG GAAACCTCTATCAACGAACGAAC
29 BACH1_56017 571 BACH1 TTTTGTGGGGTTAGCGTTC AAACGCCCTAATTCAACCGA
GAGTATTAGTTAATTGGTCGTCGGT
30 BEX1 55859 BEX1 CGATCACGTAAAAACCGTACG
TC
31 BEX1_12842 55859 BEX1 TCGGGGTTTTTATTTGGTTC AATCGTCACTCGTATCTCGCT
32 BEX1_12850 55859 BEX1 TTAGTTAATTGGTCGTCGGTTC ACTCTACAACTCCCGATCACG
33 BHMT2 55998 23743 BHMT2 TTCGTTTAAGTGTTTTGTCGAGG AACCGCTAAACTACGAACGACT BHMT2_56001 23743 BHMT2 TTAAAGAGTTGCGGGCGAG CGACTAACTCAAAATCACGAA
BIK_17885 638 BIK TTTAGAGTTCGGAGTCGGGT ATAATCCCTCTACGTCTCGCC
BIK_22360 638 BIK TTTTTGGAGTTTCGGTTTTTAC CACGAATAACCTCCGTTCG
BRCA1_JH 672 BRCA1 TCGTGGTAACGGAAAAGCGC AAATCTCAACGAACTCACGCCG
C10orf13_11683 143282 C10orf13 CGTAGGTGTATGTGTTTGGGC TCTCAAACCACGTTTATCGAA
CALCA_2 796 CALCA CGTTTTTATAGGGTTTTGGTTGGAC AAATCTCGAAACTCACCTAACGA
TTAGCGTTGGGTAAATATATGATTG
CCK 885 CCK GTTTCCAACCGAAACAACCCG
GTCGAC
CCK_13381 885 CCK TTAGTTGTCGGGTTGTTTCG GACACTTAAATCCCCCGCT
CCK_13382 885 CCK ATTGCGAGGGTTTTTAATGC CGCTAATCTTCATACCTATATCGAC
CCND2_1 894 CCN D2 GAGTTTCGGGGTTGTTTTATTC CCAACTTACGTCACCGCTT
CCND2_25209 894 CCN D2 GAAGGTAGCGTTTTTCGATG AAATAAACCCGATCCGCAA
CD34_55940 947 CD34 GCGGTATTTTGGGTTTTGC TAATATCTTCCACTCGATACGTC
CDKN1A_24717 1026 CDKN 1 A GGGAAATGTGTTTAGCGTATTAAC AAACCAACCGATCCCGAAA
CDKN2A_8472 1029 CDKN2A GTTTTGGCGAGGGTTGTTTTC ACTATTAACTCCGAACACTTAACGA
CDKN2A_8475 1029 CDKN2A GTTTTCGTGGTTTATATTTCGC AAAACGACTACTACCCTAAACGCT
CDKN2A_9696 1029 CDKN2A TTTAAGTTTTTAGGGCGTCGTT AAATAACGCTTCGATTCTCCG
CDKN2A_9700 1029 CDKN2A CGGTTGTTGTTTTAGACGTTG ATTTTCGTAATTCACATCCCG
P16_NEW1 1029 CDKN2A GAGGGTGGGGCGGATCGC GACCCCGAACCGCGACCG
CDKN2B_27345 1030 CDKN2B TTAGAAGTAATTTAGGCGCGTTC AAACCCCGTACAATAACCGA
CDKN2B_27347 1030 CDKN2B TTTTTATTTTGTTAGAGCGAGGC ACCGAACTCAAAACCGCTC
CDO1_55928 1036 CDO1 AATTTGATTTGTGTGTGTATCGC GAAACGTAAAAATATCGTCGCA
CDO1_55929 1036 CDO1 GTTTACGCGATTTTTGGGAC AAAAACCCTACGAACACGACT
CFTR_55912 1080 CFTR TCGAGAGATTATGTAGAGGTCGTT CCTCCTCTTTCGTAAACACGTA
CFTR_55913 1080 CFTR AGTTTTTCGGGGAGTCGGT GCAAATAAACGACAATCGC
CHL1_55888 10752 CHL1 GAATCGAGTGAAATTATCGGG AATCGTACTTTTCAACATCTCCG
CHL1_55889 10752 CHL1 ATTTTGATCGCGTTTCGTTT AAAATCCGCTAACATCTCTCGT
CKM_55887 1158 CKM AGTAAACGTAAAGAAAGTTTGCGA ACTAACGCCACAAACCGTAA
CNIH3_55877 1491 11 CNIH3 AAGAGTTTTTGTTTTAATCGGTTTC TACAACTATACACGCCCGCC
CNIH3_55879 1491 11 CNIH3 TTATTAAATCGTTGAGGAGAGACGA GCTAAACACTATCCGTTTACGCC
CNN1_55871 1264 CNN1 GTTGATAGTTCGTAGGTAGGGTTTC TATTCTCAACGTCAATACCGCC
GTTTTCGCGGTTTTTTAATTGGTCG
CNN3 1266 CNN3 GCCTAAACCTTCGAAAATTAACCG
TATC
CNN3_13362 1266 CNN3 TTTAGGTATCGTTCGCGTTTC AAATCTCTCGCACTTCGCTT
COL7A1_55842 1294 COL7A1 CGGTTATTAGAAGTCGTAGCGT GAACCTACCAAAACCACGAAA
COL7A1_55846 1294 COL7A1 GGGTGGTACGGTGTAGTGTTTC TAACAAAAACGCCCCGAAT
COL9A3_161 15 1299 COL9A3 TAGGTTTACGGGGGTATTTGC TCCTAACCGAACTTCCCGAAC
COL9A3_161 18 1299 COL9A3 TCGCGTAGTGTGTTTTTCGTT ATAAATCCTCGCTAACCCGAAT
COL9A3_16187 1299 COL9A3 GGGTTTATTTCGAGGCGTT TAAACGTAAAACCGCGACC
COX7A1_55819 1346 COX7A1 GTAGCGTGATAGTTGGGGAC AAAATACGAATCTAAACCGCCT
CRIP1_1 1396 CRI P 1 GTCGTTTTAGGGATTTAGCGT AATATACTTCGATAAACGCGCC
CRIP1_2 1396 CRI P 1 TTTCGTTTTAATTTTGTAGCGT ACTCGAAAATACCCGAACCGA
CRIP1_4 1396 CRI P 1 AGAGGAAACGGTAGAGATCGTAG ATAAAAACCCCTACGTCCGAAA
CSPG2_1 1462 CSPG2 TTAGAAAGGTTCGGGGTAGTTC AATCTCCTCCTCGACTCTAACG
CSPG2_23363 1462 CSPG2 TATTGTAGCGTTGCGCGATT GCATCTTCCAACACCGTCC
CST6_17991 1474 CST6 TTAGTTTTAGGTCGCGGGG ACCGTCAATACCGTCGAAA
CST6_17993 1474 CST6 GGGAAGCGTTTTGGTACGC AAACGAAACTCGAAATTACCG
CST6_55797 1474 CST6 ATGCGATTAGGGTTAGGTTTAGC GACTCTAAAAACTCCGACGACA
CTAG2 246100 CTAG 1 A GTGAGAATCGGTTACGTGTTTC GAAATCCTCAAAACGCCTACG
CXCL1_55776 2919 CXCL 1 GAATTTCGTGAGGTAGGAGGTC GAAATCATATAAATCCCGCCC
CYCLI N D2_2 894 CCN D2 GGTGTAGCGTTTAGGGTCGTC CGAATTTTTCCTACGTAACCG
CYP1A1_23831 1543 CYP1A1 TATTCGTGGTGGTGTTGAGC AAAATATAAAAATCGAACCGACC
CYP24A1_55769 1591 CYP24A1 AATTACGGTCGTCGTTGTC AAAAATCATACGAAACCCGAA
TTATTTTAGGTTCGGACGTTTTCGT
CYP24A1_bay 1591 CYP24A1 GACTAACCAAACTCCGAAAAACG
TTATTTC
CYP26A1_55745 1592 CYP26A1 CGTGGAAGAGAGTTTATTCGGT ACTCTAACCTTCCCGACGAT
CYP26A1_55746 1592 CYP26A1 GATTTGTTTATTGCGTTCGATG AAACCTCCAAACGTCTCCG
CYP26A1 55754 1592 CYP26A1 AGAGCGTATTGGTTAGTAGCGTC CTATAAAACGACAACGCCGTAA 89 DACT1_23462 51339 DACT 1 GTCGGGATAGTAGTAGTCGGC TCATACCGCCCTATAACGAAA
90 DAPK1 1612 DAPK1 GGATAGTCGGATCGAGTTAACGTC CCCTCCCAAACGCCGA
91 DDX27_55724 55661 DDX27 GTTATCGTTTTGAGGTCGGAG CTCTTAAAAATACGCATACGACA
92 DDX27_55728 55661 DDX27 TTTTCGGAGTTAGATTCGGG CTTCTACGACAACATACTTACGAAC
93 DDX43_8890 55510 DDX43 GTGTAGAGTTGGACGGTAACGA AACTTCGCCGACTAACAACGA
94 DDX43_8891 55510 DDX43 TTGCGTGAAATTAGTGCGAAG CCGTAACGTCGTAAAAATAACGTA
95 DNAJA4_8894 55466 DNAJA4 TTTTTAGTTTTATTTTTCGGCGTAG CTTACCCTTCCGTAAATCCGT
96 DNAJA4_8897 55466 DNAJ A4 TTTCGTAAGGAGACGGTGACG GTCGAAAATCTAAACCGCGAA
97 DSC3_52537 1825 DSC3 TTTACGTTTTTCGGTTGTTTC TACATTAAAATACACCGCGCA
98 DSC3_52538 1825 DSC3 TTTTTGGTTTTGTTCGGTATTTC CGACGAAATCTAACTTACCACG
99 DSC3_52554 1825 DSC3 GTCGGGTAGGGTTAGGAGAAC CTTAAAAACAAACAACGACGAAA
100 DUSP6_18075 1848 DUSP6 CGTATTTATTATGGGGGTCGAG ATTAAACCCCGCTCCGAAA
101 DUSP6_18077 1848 DUSP6 AATTTGTTTTAGTCGGTTCGTTT ACGTCTCAATAAATACATTCTCCG
102 EIF5B_55689 9669 EIF5B GGTTGTTGTTTGGTCGGTC ACGTAAAAACGCAAAACGC
103 EIF5B_55693 9669 EIF5B GGTTTAGGTTTGGTATTTCGC CGCCGTAACTCAATAAAATATACGA
104 EPB41 L3_19071 23136 EPB41 L3 GGGATAGTGGGGTTGACGC ATAAAAATCCCGACGAACGA
105 EPB41 L3_19072 23136 EPB41 L3 GCGTGGGTTTTCGTCGTAG CCCAAAACTACTCGCCGCT
106 ERCC3_2A 2071 ERCC3 GGATTTTCGTCGTTGGGTC AAAATATCCCGCCTCTAAACG
107 ERCC3_2B 2071 ERCC3 TTATGGGTAAAAGAGATCGAGCG AAACGAAAAACAAAACCGACA
108 EREG_18102 2069 EREG TTTTTGGTTTTCGGTCGTTT AAAACAACTCCGTACTTATCGAAT
109 EREG_18104 2069 EREG TTTTCGGGTTTTAACGGGG TAACGATCCTCCTACCGCTC
110 EREG_18105 2069 EREG TAGCGTAGGGATTTTGTCGGT CTACCCGTACACTCTCCGC
11 1 ESR1_2 2099 ESR1 CGGGAGTTTAGGAGTTGGC CTACCCCGAAAACCTACGA
112 ESR1_24372 2099 ESR1 GGTAGGGTGTAGATCGTGTTTTC GTCGCCTCTAACCTCGAAC
113 ESR2_55651 2100 ESR2 GATTGTAATTTGAGCGCGG AAAACCTAACAAAACGACGACA
114 ESR2_55653 2100 ESR2 TTTTTAGTTTTGGGGACGC CTATAAAATCCAACGCCCG
115 ESR2_55656 2100 ESR2 TGAGTTGTAGGAGGTGCGTTC AACACGTACTTTTCCCGCAT
116 ESR2_55657 2100 ESR2 TTTTCGTTAGGAGGTAGTTGTAAGC GAATAATCCCTCCCCGACC
117 EYA4_22346 2070 EY A4 TTAGTTTGGAGGTTGTAGTCGTTC CCCTAAATATCCCGAATAACGC
118 EYA4_22350 2070 EYA4 GAGAGAGCGCGTTTAGTCGT TCCTATCCACGTAAAATTATCGC
119 FAM20B_55630 9917 FAM20B ATATAGGGATACGCGGTTCGAG CCGTCCCCGAATAACTACG
120 FAM84A_55594 151354 FAM84A CGTAGATTTTCGTTTTTGGTTTTC GAATCCCCTATAAACAACTCGCT
121 FAM84A_55600 151354 FAM84A GTCGGTTAGTTTACGTGTTTTTATC CTATAACGACTTCTCCGCCC
122 FAM84A_55616 151354 FAM84A GTTTTGTTTTGTTTCGTTTCGTT AATACGCACACCTCCGCTC
123 FAM84A_55624 151354 FAM84A CGGAGTAGTCGGTTTTAGGGTTC CGACGAAAATTAACACAACTCG
124 FAS_18137 355 FAS TTAGCGTTTTGAGTATTCGGG TACGACGACAACGACGCAC
125 FAS_18139 355 FAS GGAGTTGTTTCGTTTGTTTAGC AACCCGAATACTCAAAACGC
126 FAS_18143 355 FAS TTTTTGATTATCGGGGTTTTTC AACCAAATCACTCGTAAACCG
127 FAS_18147 355 FAS GTGCGTCGTTGTCGTCGTA CTTACCCCGTCTTAATCCCG
128 FBLN1_54935 2192 FBLN 1 ATTTCGAGTTTTCGTGGTTTC CGACCTTACGCTACTAACGACC
129 FBLN1_54936 2192 FBLN1 AGATGAGGATCGGAGTTGGTC CGAAACCACGAAAACTCGAAA
AACGACCTCTAAAAACCGAATCAAC
130 FBLN2 2199 FBLN2 TTCGTCGGAGAGGGGGTC
G
131 FBLN2_13328 2199 FBLN2 TAGAGCGGAGGAAGTTGCG CAAATACGAACACAAAAACCGA
132 FBN2_18150 2201 FBN2 TCGGAGTTTTATAGGGTAACGAA CTCTTACTAACCGCACGCC
133 FBN2_18151 2201 FBN2 TTGGAGATTTCGATAGAGCGT AAACTACCGACTACACCTCCG
134 FKBP4_31086 2288 FKBP4 TTCGTAGGTAGCGTTTTCGTT ATCTCCTCGACTATCATCTCCG
135 FKBP4_31088 2288 FKBP4 TATTAGTTCGTTGCGTTTGCG GTAATAACGACCGATCCCGAA
136 FLJ21511_57795 80157 FLJ2151 1 TTGTATTTTCGGGGTTGTTTC CTTTACCTCCTTAACGCGAAC
137 FLJ21511_57802 80157 FLJ21511 TATTTTTATTTTGGGTACGGAGC CGAATACCAACTCCCCGAA
138 FOS_22333 2353 FOS GCGTAGGTAAGGTTGGTTTTTC CTACTAAAACGTCCCGCCC
139 FOS_22338 2353 FOS CGGGTTGTAGTTAATATCGAGG CTCTCTCATTCTACGCCGTTC
GCGATAGGTTTTTAGTAAGTAAGCG
140 FOXL2 668 FOXL2 CTCTCCGCTCCAAACGCTAACGCG
C
141 FOXL2_13395 668 FOXL2 GTTTCGTTATAGGGGCGAA CTAATACGCCAAAAACCTCGCT
142 GADD45G_57135 10912 GADD45G TATATTAGAAAGCGGGTGTCGGT GAATACGCCCCTACTACGCT
143 GADD45G 57137 10912 GADD45G GTGAGGTAGTTTTGACGTTGC GACTCCCGCTCACTACGCT GAAGTACGAAAGTTGGTTTGAGGT
144 GALE_57121 2582 GALE AAAAACCGAATAAACGACGACT
C
145 GALE_57124 2582 GALE ATTCGAGTAGGTTCGGGAGAC CGCTACCAAAAACACGACC
146 GALE_new2 2582 GALE GTTATTGGGATTTGGCGTC GCTATAACGAAAACTACGCAACG
147 GDA_57106 9615 GDA TTCGATTAGTAGATTCGCGTTG TCCCTCGAAAAATATACGCC
148 GDA_57108 9615 GDA GTATCGGTAATCGTTCGGGT CAACGAAACTCTCTACAACTACGA
149 GDF10_57718 2662 GDF10 CGGGGATATGAGTTATGGC CAATCCGCAACCTCCGATA
150 GDF10_57726 2662 GDF10 GTTATAGTCGTTCGGAGTAGCGT CGCAATAAACTACAAACTTTCGC
151 GDF10_57728 2662 GDF10 TAGTTGGGGTTCGGGTTTC CTAAACTTCGACCCTTTACCG
152 GDF10_57734 2662 GDF10 TTTTTATATTCGCGGGCGT AAAATCGTCCCTAACCCGA
153 GJB2_22303 2706 GJB2 GTATTTCGGGCGGTGTTATC ACACACGTCCTTAAACCGAA
154 GJB2_22306 2706 GJB2 TGCGGAGTATAGAGGATAACGA AACGAAAACGACTAAAAATCGAAA
155 GPNMB_52603 10457 GPNMB TTAATTTTTAGTTTTTCGATTGCG GTACTACGCGCTAACACCGA
156 GPNMB_52605 10457 GPNMB GAAGCGGTTAAAGGCGTAG TAAAAATTAAAACGCGACCG
157 GPX1_57073 2876 GPX1 GTTTAAGAAGTTAGCGGAGCGT CCTATACCACGTAACCCGC
158 GPX1_57074 2876 GPX1 GGTAATTTGGATCGTTGTCGT GCGTACTCCTCCTAAACCGAA
159 GPX7_57065 2882 GPX7 CGGGGTAAATTGGTGTCGT AACCGAACTACGAAACGTCC
160 GPX7_57070 2882 GPX7 CGGCGAAGGTTTGGATTTC ACGTAACACGACCTTAACGC
161 GREM1_29775 26585 GREM1 AGGTTTTGTATGTGACGGAGC ACGACTATTACAACCTTCCTCGT
162 GREM1_29777 26585 GREM1 GAATTTGGTACGATTTTACGGAG ATCTAAACTTTCCCTATCGACCG
163 Gst-Pi 2950 GSTP1 TTCGGGGTGTAGCGGTCGTC GCCCCAATACTAAATCACGACG
164 Gst-Pι_New1 2950 GSTP1 TCGTTATTAGTGAGTACGCGC AATAAACGAAAAACCCTACCGA
165 Gst-Pι_New3 2950 GST P 1 ATTTAGTATTGGGGCGGAGC TAACGAAAACTACGACGACGA
166 HCP5_57050 10866 HCP5 TTGTTTTCGGGAGTCGTGG AATTTACTCCTCCGAACGAAC
167 HHIP_23319 64399 HHIP AGTAGTAGGAATAGAAACGGCGA AAAACTACAACCGCCGACA
168 HIST1 H3G_57040 8355 HIST1 H3G CGATTTTTCGTATTAGGCGTTG AAAACCTCATCGCTACCGTC
169 HIST1 H3G_57042 8355 HIST1 H3G CGGGTGAAAGTAGGCGGTT AAACATTCAACTCGCTCGC
170 HMG20B_57022 10362 HMG20B TTTTTATGAGCGTTTAGGCG TACCTCCGAACTTTTACCCGA
171 HMG20B_57023 10362 HMG20B GAGTCGGTTGAGTAGCGTTG AAACTCGAATCTTCTAACGAAA
172 HMG20B_57033 10362 HMG20B TTATCGCGGGGTATTTTAGTTC AAACCGTAAAACATAACCGCTC
173 HMG20B_57034 10362 HMG20B GGTGAAAATAGTCGCGGAA CCCGAAAAACCTAAACCGA
174 HORMAD1_57018 84072 HORMAD1 GGTTAAAAAGTTATTGTCGCGTT TACACGAAACTACACGCGCT
175 HORMAD1_57019 84072 HORMAD1 TATTGTTTGAGACGTGGCGTT ATCGAAATCACCTTTCCCG
176 HOXA10_57000 3206 HOXA10 GTTGTTTTATTGCGTTTGTCGT CGAAATCTACCTACCGCCC
177 HOXA10_57002 3206 HOXA10 TTTATTCGGTAAGATCGGGG AATCTAAATCCCGAAACGCA
178 HOXA4_56994 3201 HOXA4 GGACGTGGTTCGTATGTAGGTC CCCTATAACTACCGCGACGA
179 HOXA5_1 3202 HOXA5 GTAGTTCGGGTTATTTGGATAGC AAAAATACTATAAACGCACAAACGA
180 HOXA5_2 3202 HOXA5 TTGTCGGAGTGTATGTTCGTC CGCTATCCAAATAACCCGAA
181 HOXA9_1 3205 HOXA9 GGATTCGTTTTTGTTGGGC ATCGAAATACTCGACCAACGTC
182 HOXA9_2 3205 HOXA9 GTTGCGGAGAAAGATACGAG CGAACGAAACATCACGACC
183 HOXA9_9564 3205 HOXA9 TAGTTTTTAGTTTAAGGCGACGG TAATAATAATACACCGCAACGAA
184 HOXD1_13247 3231 HOXD1 AGGTCGCGTAATTTATTTGGTC TCCCGAACGTACTAAAACGAA
185 HOXD1_13254 3231 HOXD1 TAGCGTTAAGTAAATCGAAAGCG CGATAAAAACTACTCTTCGCCC
186 HOXD1_13257 3231 HOXD1 TTCGAGAAGACGGTCGAGG ACTACGACTTCCTAAAATCCGAAC
187 HOXD1_56977 3231 HOXD1 TATGTAGTAGTAGCGGCGGG CTACAAAACCACGAACCGAA
188 HOXD1 (2) 3231 HOXD1 GTCGGTTGACGTTTTGAGATAAGTC ACCGTCTTCTCGAACGACG
189 HUS1 B 135458 HUS1 B TGGTGTACGATTTGTTCGTGC TCCAACGCGACAAACG
190 HUS1 B_12677 135458 HUS1 B GTTGGGCGGTAGGTAGTTTC AACTAATCTCGTCCCTAAACCG
191 HUS1 B_12680 135458 HUS1 B GTCGTTTAGTTTGCGCGTT GTAACTACCCACGTTCGCC
192 ICAM1 3383 ICAM1 TAAAGACGTTTTCGCGGTTAAGGTC ACCACGTCCGAAAAAATCGACG
193 ICAM 1_13230 3383 ICAM1 TAAGTTTAGTTTGGTCGGGAAAC CTAAAACAATCGAACGACGATA
CTAACAAAATACCGAAATACGAAAAA
194 ICAM5 7087 ICAM5 CGTTTCGGTTTCGTGTTTTTTATC
AATACG
195 ID4_56952 3400 ID4 GCGAGTAGGGTTTAGGCGTT AAAACTACGAAAATATACGACCGA
196 ID4_56956 3400 ID4 GTTATTTAGGTTGTGGTCGTGTTC ACGATAAACCCGATACGCC
197 IGSF4_18983 23705 IGSF4 GATATGGCGAGTGTAGTGTTGTC AACAAAAACCGAAACCGAA
198 IGSF4 18987 23705 IGSF4 TCGGATTTCGTTTTTAGCGTAT GAACACCTACCTCAAACTAACGAC 199 IL17RD_54549 54756 IL17RD TAGGCGTTGATCGTAAAGAAGAC CGAAACTAACCAACGACGAAC
200 IL17RD_54550 54756 IL17RD TTTAGTTATAGGTGTCGGCGTTT CAACTCTACTCCGTCTTCTTTACG
201 IL17RD_54556 54756 IL17RD TTTTTGTCGTCGTTAATTCGC AAATAAAACCCAAACCGACCG
202 ING1_24659 3621 ING1 GTTTATTTGCGTTGCGTTC CTCTAACACCCGAAACGACC
203 ING1_24663 3621 ING1 GACGAAGAAGGGTTAGAGGAAC CCTAAAACTATAAAACGCCGAAA
204 ING1_24679 3621 ING1 TAGGTAGTTAAGCGTGGCGAA ACTCTCTAATTCCGAAACGACC
205 ING1_24685 3621 ING1 TCGAAATAGAATTGGTAATCGTAG CGCCTCCTCTTCATCGTAA
206 INHBB_56906 3625 INHBB GTATTTCGTGTCGCGGTTC CCGAATACAATACCCTCGCT
207 INHBB_56909 3625 INHBB CGTTCGTATTTGAGCGTTTC CGAAAACGACCTACCCGTCT
208 IRF7_18344 3665 IRF7 GTTAGGTCGCGGTTATAGGTC CATACCGAAACAACGTCGATA
209 IRF7_18346 3665 IRF7 AGTTGAGAATCGGACGGGG AACGAATCAAACTCCCGAAA
210 ITIH5_56885 80760 ITIH5 TCGTGATAGTGAGTGAGTTTTTAC AACGAACACGTCTACAAAACGA
211 ITIH5_56887 80760 ITIH5 CGTTGTAAAGCGTGTTTCGT CTACGCCTCTTCCTACGACC
212 KCNG3_56878 170850 KCNG3 GAGGGTTGCGTATATCGAGG ACGAAAAACTATTCCGCCC
213 KCNG3_56880 170850 KCNG3 CGTTGTTAGTAGCGCGTTTTTC CAACCGATAAAACTCAACGC
214 KIF1A_1 547 KIF1A TTTTTATTTTTCGCGGGGTTC AATCGCCAACTAATTTATCGC
215 KIF1A_2 547 KIF1A GAGGTTTTAGGGGTACGTCGG AAACCGAAACCGAACTATAACG
216 KL_56871 9365 KL GAATGAATTTGAGCGTTTACGA AAATACGACAAACGTCGCCC
217 KL_56874 9365 KL AGATGGACGTATTTTTGTCGTG GACGACTTCCTCTAAACCGTAA
218 KLF4_2 9314 KLF4 ATTAGTAAGGCGAGTAAGTAGGTTC CCCACGACAACACTCGAAA
219 KLF4_24636 9314 KLF4 TTTGTTTAATTGCGTGTGAGC ATATACCAATTCCGAAAACGTCC
220 KLF4_24642 9314 KLF4 TTTTCGGGATAGTTTTCGTGT TAATAAAAACCCGACTTACGCCC
221 KRT14_56851 3861 KRT 14 TTGTTGAAGTTATCGTTATAGTCGT GACCTATCTATCTCATCCTCCCG
222 KRT14_56852 3861 KRT 14 GTTAAGTTTAGTGCGCGGG TTTCTTATCCCGCCAAACG
223 KRTCAP3_56842 200634 KRTCAP3 GTTGATGCGTGTGGGTTTC AAAATTAACCACGTACCGCAA
224 KRTCAP3_56843 200634 KRTCAP3 TACGGGTATTAGGGGAGTCG AACAACAACTAAAACGAATCGAA
225 LEPR_56815 3953 LEPR TATATTTTAAGGGACGAAGGGAGC CTAAAACTACGCCTTCCGC
226 LEPR_56822 3953 LEPR TTTGGAGTCGTAGGCGTTG ATTCAAACCCGACCTCGCT
227 LEPR_56824 3953 LEPR TTAAGTTAGATCGGGAGTCGTTT ACTAAAATCGAAATTCTACACCGA
228 LGR6 59352 LGR6 GTAGGGGTACGGTTAGGC GCACGTACCCGAAAACTAAACG
229 LGR6_56719 59352 LGR6 TAGATATTGCGTAGTAGGCGGG CGCCTTTCAAAAACTTCCG
230 LGR6_56730 59352 LGR6 TTTTGATTGTATCGTTTCGGC ATCCCGACGAACTACACCG
231 LGR6_56731 59352 LGR6 GTTCGTAGGATTAGGCGGG CTCGAACTATCCGCCGTTC
232 LHX6_23427 26468 LHX6 CGTTCGTTATGGTGAGTTCGT CGTAAACAACAATTCGTAACCG
233 LHX6_23428 26468 LHX6 GTTTTGTCGTTGGAGTTTCGT TCTCTCTAAATCTCTACCTCGTCGT
234 LHX6_23430 26468 LHX6 TTGGGTTTCGGATTTTGTTC AACTACCGACTACCGACCGA
235 LIPG_56689 9388 LIPG CGTTGTATATTTATCGCGTGGAC CTAAAATCGCAACTACCTACGAAA
236 LIPG_56693 9388 LIPG ATTTGGCGGTACGGAGTTC CTCAAAACGAAAACAAATCTCG
237 LOX_18977 4015 LOX GGTAGAGGCGAGGAGTTGTTC TACACAAACCGTTCTAACCCGA
238 LOX_23395 4015 LOX GTTAGATTGATTTCGTTCGAGG AACTAAAATACCCGTACTCCGCT
239 LSMD1_56678 84316 LSMD1 GTTATATTTCGCGTAGGATTCGG ACAACGCCTCTAACTACTATCGAAC
240 LSMD1_56679 84316 LSMD1 GAGGTTTTCGGGTAAATGGTC CACTAAAACTACTCTAACGCCGA
241 LSMD1_56683 84316 LSMD1 ATTTTTATATGTTTTACGCGGGA TATCCTTTTCCGACCACCG
242 LTB4R_31247 1241 LTB4R GTAGTTCGGTTTTGGTTCGTC GATCCCTCCACAAATAACGAT
243 LTB4R_31250 1241 LTB4R TAGTAGATTTTTAGCGGTGAAGACG AAAACCTTAACGAAACTAAACGAAA
244 LTB4R_31257 1241 LTB4R TTTAGTTCGGTTGAGTTAGAGGC CAAATAACAAATAAACGACGCAC
245 LTB4R_31261 1241 LTB4R TTTTAATGTTGATAGCGTTCGG CTAACCGCCTCCGAATAAACG
GCGGGGTTTTTTTTATCGGTTAGAT CAACGATACCCAAAAAAAATCAACG
246 LY6K 54742 LY6K
TC CG
247 LY6K_12884 54742 LY6K TTTATCGGTTAGATTCGGGG AACGAACAACGCCATCGTC
248 LY6K_12885 54742 LY6K GCGTATTAGTGTGTGAGGTTACG TACAAAATTACTCACGAACGACC
249 LZTFL1_56661 54585 LZTFL1 GTCGGTTATTTATTATGGCGGT ACGATCCCAAACTTAACGAAA
250 LZTFL1_56669 54585 LZTFL 1 ATGCGTATGTGGTAGTTAATCGG CGAAAACCGAAACACGTAA
CGAGAGTCGTAGAAGAGAATCGTA
251 MAGEH1_57803 28986 MAGEH1 CGCTACCACAAAAACGACC
A
252 MAGEH1_57810 28986 MAGEH1 GTCGTTTTCGGGGGTGTTC AAAACCGCAACAATCGCAA
253 MAL 1 4118 MAL TTCGGGTTTTTTTGTTTTTAATTC GAAAACCATAACGACGTACTAACG 254 MAL_56656 4118 MAL GTGGCGGTGGTTTAGTTTC TTCATTTTTCCGCTAAATACGTT
255 MAL_56660 4118 MAL TTTTGGTTTAGTTTTCGTTTTGC GTACGCTCTCTCGAACGCC
256 MET_54299 4233 MET GTTAGTTTTTATTTTAGCGGTTTTC CCCAACACGTATCTATCTACCTCG
257 MET_54300 4233 MET TAGCGGCGTAAGGATTATACG CGAACCAAATACGAAACGACA
258 MGC33846_56643 220382 MGC33846 GTTTATGAGGTCGTTGTAGCGT AACAAACCGAACAAATCGAAA
259 MGC33846_56649 220382 MGC33846 GGTAGCGTTGCGGATAGTTC CCGAAACCGAAAAACACGA
260 MMP2 4313 MMP2 TATCGAGTTAGCGATTTTCGGGC CGCCCTCAAAAAACCCGTAAACG
261 MMP2_52612 4313 MMP2 GGGTCGGATTATGAGTCGTT AATTAAAACCTACTCCGCGAC
262 MYCL1_5421 1 4610 MYCL 1 GCGTTCGTGTTTTGGTTATTC ATACTAACTACCGAACTACCCCG
263 MYCL1_54212 4610 MYCL 1 GATTCGATTGTGGGTAGCGAG AAACTACCTTCTAAACACGACGC
264 MYCL1_54213 4610 MYCL 1 GTAGTTTGCGTTTAGTTTTCGC TAAACTCGCTACCCACAATCG
265 NAALAD2_56618 10003 NAALAD2 TTTGCGAAGGTTAGCGGAG CTTAAAAACCGAACTTAAAACGC
266 NAALAD2_56623 10003 NAALAD2 AAGGTATAGACGGTTTTTGGATTTC GCTCTCTATTTCTCTACAACCCCG
267 NAGS_57828 162417 NAGS GGGTGTCGAGTTTTAGGTTTTC TAAATATCCCGCTACACCAACGA
268 NAGS_57831 162417 NAGS GGATTCGGTATTTACGACGG ACTCAACACCGCCTAATCG
269 NDP_1 4693 NDP TTTCGTGGAAGGGAGTCGAG CGCACTAACAAATAAAAATACCACG
270 NDP_8597 4693 NDP GTTACGGAAATGTGAGGCGTTC CTATTCCCATCGTAAAATACTCCGT
271 NDRG2_56603 57447 NDRG2 AGATTTTGTGGTTTCGTCGTT ATCCCCCGAACATTACGATT
272 NDRG2_56609 57447 NDRG2 TATCGAGAGGGACGCGGTA ACCCTATAACTTCGCCGCT
273 NEDD4L_56593 23327 NEDD4L AGAGAGGTAGGTATTCGCGGT GCCCAAATAAAACCCTACG
274 NEDD4L_56595 23327 NEDD4L ATAATAGTAACGGCGGCGAG GATACCTCCCCAACACGACA
275 NEDD4L_56599 23327 NEDD4L CGTGGGGTTGAATTTCGTT GAAAATACCCTCCCCGTTC
276 NEDD4L_56600 23327 NEDD4L TTGTAGACGTTGGGTACGAG CTACTCGAAACTCCCCGAAA
277 NEF3_13215 4741 NEF3 TGAGTTATACGTTGGATTCGTTG AAAAACCGCTAACGCGACT
278 NEF3_13218 4741 NEF3 ATTATTGGGTCGGGGAAGC TCCCGATTACGTTTCGCAC
279 NEF3_13224 4741 NEF3 TATTATTTTTAGGGTGGCGCGTA AAAATCCAAACGCTACGACA
280 NEFH_18450 4744 NEFH TATGTCGAGGTAGAGAAATCGG CGAAACCCTATCACAAACGAA
281 N EFhM 8452 4744 NEFH GTCGGATGAAGTATTCGGG CCCTACAAACGACGACGAAC
AAAATCGCTAACGTAAACGTTCGAC
282 NEURL 9148 NEURL GAGCGTTTAGAACGTTTCGCGTTTC
G
283 NEURL_13058 9148 NEURL GTTGTTATGGTGATAGTTGCGTC GCCGAAAACTTACGACTACGA
284 NEURL_56560 9148 NEURL CGGTTTTGTTATGTCGGTTTTC ACTAATCACCGAATCGCCC
285 NEURL_56564 9148 NEURL AGGTATTCGCGGGGTTTTATC AATAACTACACTACCCGTCGCC
286 NPPB_13212 4879 NPPB GGAGTACGGGGTGATTTATAGC CGACGAACAAATACTACGCTACG
287 NPPB_13214 4879 NPPB TAGGGTGTATAGCGGCGAG ACAACGAAAACAACCCCGA
GACGTAAAAAATAAAAACCGAACAA
288 NPPB(2) 4879 NPPB GGGGATTTGTTTGTGTTTTTAGCGC
AACCG
289 NPTX2_57773 4885 NPTX2 TTTTAGTTTGTGACGTTCGCGTT TAAAACTCTCGAAAACCTCGACT
290 NPTX2_57779 4885 NPTX2 GCGTCGTTTTGTATGGGTATC CCCGATAACCGCTTCGTAT
291 OGDHL 55753 OGDHL TCGTTAGTATCGTGGATAGC TACAAATCAAAAAACTACGCG
292 OGDHL_19631 55753 OGDHL GTTTTAGTTTCGTTTTGCGGTT GCTCCTAACGCTATCCACG
293 P14 1029 CDKN2A GTGTTAAAGGGCGGCGTAGC AAAACCCTCACTCGCGACGA
294 P16 1029 CDKN2A TTATTAGAGGGTGGGGCGGATCGC GACCCCGAACCGCGACCGTAA
295 PAX3_18479 5077 PAX3 TTTGGGTATAGCGTCGGTT ATTCCCGAAAATCATCCGC
296 PAX3_18482 5077 PAX3 TTTGGGTAAGGGGCGTAGC CCCGAATAACGAAAACACG
297 PCSK6_56480 5046 PCSK6 TCGGGTCGTTTAGTATTTGTATC CTAACGCTACCTACCGCCT
298 PCSK6_56483 5046 PCSK6 ATAGGATTGCGGAGGCGTT ACCAAATAAATACGACCGACC
299 PDLI M3_18531 27295 PDLIM3 GGAGGTATTCGAGTTTGGTAGTTTC CATCCTCTAAATCCGCAACG
300 PDLIM3_18532 27295 PDLIM3 AGGTGTGATTTTAAGAGAGCGAC GAAAATACGCAACCCGACC
301 PIK3CA_66163 5290 PIK3CA AAAAATAGGGGCGACGGAG GACACCGAAACTACCGCTT
302 PIK3CA_66165 5290 PIK3CA ATAGAGTTATCGCGGTCGGG CTCTAACTACCGCCTCGCTC
303 PLAGL1_52944 5325 PLAGL1 CGTTCGTTCGTTTAGTATTCGTT ATTTATCATTCAACCCGTCGAT
304 PLAGL1_52949 5325 PLAGL1 CGTTGTAGATTTTAGGTCGGTTC CTAAACGACACCCACACGTCC
305 PLAU_18620 5328 PLAU GTAGTATAGTGCGGAGATCGTAG AAATCTCAAAACCGCGACA
306 PLALM 8623 5328 PLAU TAGGGTAAGAGGCGGAGGAAC AAAACCTAATCACCGCGAAC
307 PLXNA4B_57788 91584 PLXNA4B CGTTCGTTCGGTTTTATTTTTC CATCTACAATATCAATACGACGCT
308 PLXNA4B 57794 91584 PLXNA4B TTGGTATTAAGGATATTTAGTTCGC GACTCTCGAAAACTACGATAACGAC 309 PNMA3_56433 29944 PNMA3 TTTTTCGTTTCGTTGGTATTTTC CTATAACGACCAATCCCCGT
310 PNMA3_56439 29944 PNMA3 TTTGTGAATGCGAGTAGGGC ACTACAAAAACGCCGCACG
31 1 PON2_56425 5445 PON2 TAGGTTTAGGGCGGAGTTTTC ACGATACCATCTTCCCGACT
312 POU2AF1_23443 5450 POU2AF1 TTGTTATTTTATTGTGCGCGTT CAAACTATAACTACCCGCGCT
313 POU2AF1_23446 5450 POU2AF1 TTTTGGGAATCGTATTTTTCGT GTTAATCTTTACACGACGCCC
314 PPP1 R13B_23470 23368 PPP1 R13B GTTCGGTTTCGTATCGGTT AATAACGACGACTTCCTCCG
315 PPP1 R13B_56416 23368 PPP1 R13B GTTCGTCGTAGTTGTCGTTTC CGAACTATACCCACCGCTC
316 PPP1 R14A_8724 94274 PPP1 R14A CGTTATCGTTAGAGGGCGTAG CTACATCGAAAACTCCCGAAC
317 PPP1 R14A_8725 94274 PPP1 R14A CGTTATTGTATTTTAGTTTGGGC ACGATCACTTATTCCTTCGTTC
318 PROX1_56401 5629 PROX1 TTTTTCGGTAGAGAGAGGAAAATAC TATCGCACAAACTCACCGTC
319 PTEN_10192 5728 PTEN GAAGAGGAGGCGAGAAACGTC CTCAACGCCTATAAACAACCG
320 PTEN_9406 5728 PTEN TAAACGAGTCGAGTTATCGGG CAACCTACTATTATATCGCCAACG
321 PTPRD_56390 5789 PTPRD TTTTTAGTATTCGTGCGGAG GAAAACAATAATTCCAAACTACTCG
322 PYCARD_52546 29108 PYCARD GAATAGAAAGCGGAAGAGTTCG AATAACCGAACAACTACAAACGAC
323 PYCARD_56380 29108 PYCARD AGTCGTCGATTAGGAGGAAGTC ACAACGAACCGAAACGAAA
GGGATTAGAATTTTTTTATGCGAGT
324 RAR2β 5915 RARB TACCCCGACGATACCCAAAC
TGT
325 RARB_23618 5915 RARB TTTTAGTAGGATTTATTTGTCGGC GTTACTCTTTCCACCTATCATCGAA
326 RARRES1_57577 5918 RARRES 1 GTCGTTTGTTGTAGGAGTTTGC CTACTACTATTACTCGCCCCGAT
327 RARRES1_57581 5918 RARRES 1 GCGAAATCGTAGGGGAAAC ATAAAAACGCCTCCCCGAAA
328 RARRES2_57694 5919 RARRES2 TTGGGATCGGAGAGCGATA AAAAACAACTACGCAAACGAAA
329 RARRES2_57700 5919 RARRES2 AGTAGAGTAGCGGAGAAGAGGTTC GACTACAAACGACTCGCCC
330 RASSF1A 11 186 RASSF1 GCGTTGAAGTCGGGGTTC CCCGTACTTCGCTAACTTTAAACG
331 RASSF1_8476 11 186 RASSF1 TCGTTTTTAGGAATGATTTTATCG CACTCTTATACGCTTACCCGAAC
332 RASSF1_8480 11 186 RASSF1 GTTTGCGAGTTAGCGAGGTTC TAACCTAAAACAACACGACGA
333 REC8L1_13013 9985 REC8L1 GCGTTTTTGTTGTTAGGTTTGC AATAAACTCCGATTCTCGCCT
CGATTTAGTTTTGTAGTAGGCGGTT
334 REC8L1 (3) 9985 REC8L1 GATCCTCGAACGCTAAAAACG
C
335 RP11-450P7 3_56363 257240 RP11 -450P7 3 CGTGTGAGTTAGGTTAAAGCG TATAAACGCTACTTAAAACGCCA
336 RP11-450P7 3_56369 257240 RP11 -450P7 3 TTATCGTCGTTTTTGGCGT ACTTAATTTCCACGACTACCGA
337 RP11-450P7 3_56371 257240 RP11 -450P7 3 TTTTTAGTGAATTTCGGTTTTC CGAATAAAACGACCCGAAC
338 RPL10_56355 6134 RPL10 GTCGTTCGGAAATAAGATAGAGAAC CTACAAACTCACCGCATTTTCG
339 RPL10_56356 6134 RPL10 TTTTTCGGGTTGATAAAGGAAC CCTAAACTTACACGAATCTCGAAC
340 RPL10_56361 6134 RPL10 AGATTTATTAACGGGCGGG AATCTATTCTCGTCTTCCGTTC
341 RPRC1_56316 55700 RPRC1 TTGGGGTTATAGGCGCGTT TCAACCTAAACAACGTAACGAAA
342 RPRC1_56318 55700 RPRC1 TTTTTACGGTTTTTACGGTG ACGATACTACTCTCCCATACGCT
343 RPRC1_56320 55700 RPRC1 GCGTTAGGTTGGTTTTTGTTC CCTTCCTTACTCGAAATCGC
344 RPRC1_56335 55700 RPRC1 GAGGTTAGGTTTAGTTCGGGC CCGAAATCTCTCGACTACGAT
345 RPRC1_56336 55700 RPRC1 TTTTTATTTAGTTTTTAGGCGTTGC ACCGAACGAACTCCACGAC
346 RUNX3_1 1669 864 RUNX3 TTTTAGAGCGGGGTATGGTATC CTAAACTATTATTACTCACCGCGAA
347 RUNX3_2 864 RUNX3 TATTTATTTTGAAGGCGACGG TAACGAACCACGCAAACGA
348 RUNX3_23117 864 RUNX3 TTATAGGATGCGAGAAGTTTGTTC AAAAACCGTAACATCACGACC
349 SALL4_12833 57167 SALL4 GAGGCGTAAGTAGGCGAAA CGCATCTACAAACTCCGAAA
350 HIN1_1 92304 SCGB3A1 TAGGGAAGGGGGTACGGGTTT CGCTCACGACCGTACCCTAA
351 HIN1_3 92304 SCGB3A1 GAAGTTGGTTAGGGTACGGTC AACTTCTTATACCCGATCCTCG
352 SCN1 B_56293 6324 SCN 1 B TTATTCGGAGAGGTCGGTG CGAAATAAATCACCGCGAAA
353 SCN1 B_56297 6324 SCN1 B GGGTTTAGTTTCGGGGTTC TAACGAATATACCCGACTACGC
354 SEMA3B_56291 7869 SEMA3B GTATCGTTCGTCGTGTCGT TCCCGCCTAACTACCGTCT
355 SESN1_29753 27244 SESN 1 TTTTAGATAGGGCGGGTTTTC AAAAACGACTCACGATACGCAA
356 SESN1_29754 27244 SESN 1 ATTAGGACGAGGTATTTGGGC AAAAACAACGTAACCTCCGTAT
357 SFN 2810 SFN TGGTAGTTTTTATGAAAGGCGTC CCTCTAACCGCCCACCACG
358 SFRP1_9378 6422 SFRP1 GGGAACGGTATTGATTGTTCGT GCTCACAAAATACAAATACATCCG
359 SFRP1_9379 6422 SFRP1 ATGTTTATGTCGGTTTTGCGT ACTACGCCTTTTATCCCCGAA
360 SLC22A3_56237 6581 SLC22A3 ATGGGCGTAGGAGGTTTTC GACCCGAAATACAACCCGA
361 SLC22A3_56240 6581 SLC22A3 TTTTTCGGGTTTTAGTCGTAG ACGTAATCTTCCTAAACACGCA
362 SLC2A14_56224 144195 SLC2A14 TGAGTTTAGCGAGATTACGAGG ATAAAACTACAAACTTCCGCCGT
363 SLC35A5 56220 55032 SLC35A5 TAGTGTTACGTGAGGTTTCGGT CTCTACGAACTATCTATCCTCGCT 364 SLC35A5_56222 55032 SLC35A5 CGGATTCGATATTTATTGCGTAG CACTAAACTCTCCCGACACGTA
365 SLIT1_23641 6585 SLIT1 TTTTCGTTATTAAGAGGTGTTTCG CGCTACGCTAACTACAAACGTCC
366 SLIT1_23643 6585 SLIT1 CGTTAGGGTTGGGAACGAT GATTAAATAACTCGACCGCCC
367 SLIT1_23651 6585 SLIT1 GCGTTATGGTGTTTTTATAGCGT TCTTCGATAACTCTACCCCGA
368 SLIT1_23653 6585 SLIT1 TTGTAGGCGGTTTGTAGTCGT GACAATCATCCATCAATCGAAA
369 SLIT2_23672 9353 SLIT2 GAGGATCGGTTTAGGTTGC CAATTCTAAAAACGCACGACT
370 SLIT2_23676 9353 SLIT2 AGGGGAAGACGAAGAGCGT CACGAACTAACGCTACGCAA
371 SLIT2_23678 9353 SLIT2 CGGTTTGTTTGGTTTTTAAGTTC ACTAATCAACACTCCGACGAAA
372 SLIT2_23681 9353 SLIT2 TAGCGGAGAGGAGATTACGC GACCCCTACATCTTAACAACCG
373 SOCS3_53602 9021 SOCS3 GCGTAGTTTTAGGAATCGGGG ACCGAAACGACAACAACGAAA
374 SOCS3_53604 9021 SOCS3 GGAGGGGTTTCGTTTAGTTTC AAAATCTCCGACTTCCATCCG
375 SOX9_29341 6662 SOX9 TGTAAGTGCGGGTATTGGTTC ACTCAAAAACTACGACTAAACGCT
376 SOX9_56211 6662 SOX9 TGAATTGGTTATTTCGCGTTT CCTCGACTACCCGAATACGA
377 SOX9_56213 6662 SOX9 GTAGTTAAAGGGCGGACGG TCTAACATTCCGAAAATACGACA
378 SPFH2_55669 11 160 SPFH2 GGACGAGTACGGAGCGTTT AAATAACGAATCAACCCCGA
379 SPFH2_55675 11 160 SPFH2 AGGTTTAACGTAATCGGGG AAAAACGCAACTACAAATCG
380 SYK_53559 6850 SYK ATTCGGGTATTTTAGGGCGTT GCTATACCTAATCCCAATCCCG
381 SYK_57767 6850 SYK GTATGAAAGTAGCGCGTCGT GCAAATAAATATAACCGCGTCC
382 SYN E 1 23345 SYN E 1 GTTGGGTTTTCGTAGTTTTGTAGATCGC CTACGCCCAAACTCGACG
383 TAC1_56185 6863 TAC 1 ATTTGTAGACGGAAGTAGGTCG CGAAACGATAACTCGTCGATA
384 TAC1_56187 6863 TAC 1 GGGTATTTATTGCGACGGAT CCGACGACAACTACCGAAA
CAAACCCACAAACGATCCGAATAAT
385 TCL 1 A 81 15 TCL 1 A GACGTTATGGTCGAGTGTTCGATATTC
CG
386 TCL1A_13087 8115 TCL1A CGATGGTTAAGGGTAGTTAGGC CGAATACCCGACACTCGAAA
387 TEX14_56176 56155 TEX14 TTGTGGGTTAGTTTTTAACGGTC CGAAATACGACTCCCGAAA
388 TF_56164 7018 TF CGAGTTCGATTGTGTTCGTT CGACTCTACGATACTACCCCGT
389 TF_56170 7018 TF TAGGTTTTTAGTTTGAGCGCGG AATAACACCGCAAAATCGCTA
390 TFPI2 7980 TFPI2 GTTCGTTGGGTAAGGCGTTC CATAAAACGAACACCCGAACCG
391 TFPI2_12620 7980 TFPI2 CGGGGTGATAGTTTTCGTG CGACTTTCTACTCCAAACGACC
392 THBS1_57460 7057 THBS1 CGAAAGTTTTTGCGTTATTTC GACGCTCGTACTCTTACGCCA
393 THBS1_57463 7057 THBS1 GGGCGATTTATTTGTGTGTATC AAAACAACCTCTACTCCGAACG
394 THBS2_18771 7058 THBS2 GGATCGAGTTAGAAAGGCGTAG ATATAAAACCGCGCTACCCGA
395 TIMP3 7078 TIMP3 GCGTCGGAGGTTAAGGTTGTT CTCTCCAAAATTACCGTACGCG
396 TNFRSF11 B_30721 4982 TNFRSF11 B TATCGGGTTGAGGAATAAGGC AAAAACTAACCGCTAACGAAA
397 TNFRSF11 B_30722 4982 TNFRSF11 B TGGTTTAGGGATTTATTACGAGC AACTACGAAAACGCACCGAAA
398 TRPV2_18803 51393 TRPV2 TTATTTCGTAGGTTGAGGTTAGGGC TCCTCTACTATCAACGCCGAC
399 TSLC1 23705 IGSF4 GGTGAGTGACGGAAATTTGTAACG ACAATCGCTATATCAAACCGACG
400 TSLC1_1 23705 IGSF4 ATTGGTTTGTTCGGATTTCGT AACACTACACTCGCCATATCG
401 TSPYL6 388951 TSPYL6 TCGTTTGGAGTTAATCGTGTTTTTATCGTTTC CCATCTACGAAATCCTAAAACGCG
402 TSPYL6_12624 388951 TSPYL6 TTCGAGAAAAGAGTAAGGCGA CTCCTCTAAAAACCGAAACGA
403 TSPYL6_12626 388951 TSPYL6 TTATTCGTTATTTTGTGTGAGGAAC ACGAAACTATAAAAACTCTCCGAA
404 TWIST1_3 7291 TWIST1 GTTAGGGTTCGGGGGCGTTGTT CCGTCGCCTTCCTCCGACGAA
405 TWIST1_9329 7291 TWIST1 TTTAGTTCGTTAGTTTCGTCGGT TACTACTACGCCGCTTACGTCC
406 UCHL1_13107 7345 UCHL1 TAGGTTTTATAGTGCGTTTGGTC CATCGACTTAAACTACATCTTCGC
407 UCHL1_57523 7345 UCHL1 TTGTTAGTAGTCGGAATCGGTG AAACAAAATAAATAACGTCTCGC
408 WIF1_23405 11 197 WIF1 ATGTTTTAGAGTTAGAGCGCGG AATTTTCCCGACAACGAAA
409 WIF1_9096 11 197 WIF1 GCGTCGTTAGATATTTTGTTGC TAACACCCAAACCGAAAAACG
410 WT1_1 7490 WT 1 TGTGTTATATCGGTTAGTTGAGAGC CGCTACTCCTTAAAAACGCC
41 1 WT1_2 7490 WT 1 GGATATACGTGGAAGTCGGG GAAACGACAACCCAAACGC
412 ZAP70_57514 7535 ZAP70 GCGGAGAGTGTGAGCGGTA AAAATCGAAACTTACAAAACCGA
413 ZIC1_57510 7545 ZIC1 GGGCGGGTTAATGAGTTGC AAAATATCACGTACTACCGACGC
414 ZIC1_57513 7545 ZIC1 GTTAGGTAGATTTTGTCGGTTCG CCCCGAACAACCTTAACGC
415 ZMYM5_57502 9205 ZMYM5 ATTTGGGGATTTCGGTTTC CTCTCCTACCGCATAATAACGA
416 ZMYM5_57506 9205 ZMYM5 GGGTGTGGTTCGTATTTGAC CCCATTAACTCCGACGACC
417 ZNF195_57489 7748 ZNF195 TTTTGATTCGGGGAGACGA CGCATCCCTACAAAACGAC
418 ZNF195_57492 7748 ZNF195 ATTTAGCGGGGTTTTTGTTTC TACGAATCACACGACCTACGA
419 ZNF365 57483 22891 ZNF365 TAGTGTAATTTTGAGTCGGATCG AACTACCACCTCCGCGAAA 420 ZNF365_57485 22891 ZNF365 TTTTTCGTTGCGTGTTAGTTTAC AAAAACCGACTTCCCGACC
421 ZNF432_57478 9668 ZNF432 TTTATGGAGAGCGGAATGC CCCAAAACGACGAAACGAC
422 ZNF432_57479 9668 ZNF432 TTTAGTTTTTAAGAGGTCGCGG ATATAACGCCACCAACAACG
423 ZNF655 79027 ZNF655 TTATCGAGAAGCGTCGGTTTC ACCGAAAAAAAAAACGAACCTAACCG
424 ZNF655 12792 79027 ZNF655 GTCGTAGTTATTGTTGGTATAACGG TCGCAACTTTTCCACGTCT
Table IB: Amplicon details (converted sequences issuing from the methylated version of the DNA )
Nr. Assay Name GenelD Official Gene Amplicon Sequence (converted) SEQ ID NO'S 849-1272 Symbol
1 ABHD3_11682 171586 ABHD3 GATATTCGTCGTAGAGCGGAATCGTTTAAGGTTATTGGGGTTGTAGGTG CGGGGTTTTAATTAGCGTTGTCGATTCGTTAGTTTTCGGTTTATTTTTG 2 ABHD3_55542 171586 ABHD3 GGTTTTGCGTTATTTTCGGTTTATTTTGGTAATGTTGTTTAGGTAGTAGAA GGTATAAGCGACGTTGAAGTTTAGGATAAG 3 ACP5_55536 54 ACP5 TTTTGTATTTGCGGTCGGTTTGATTTGTTTTTTATTTTTTTGTTTGTTTTTTT CGTGTTGGGCGTCGTCGCGATACGTTTCGCGGATATTTAGGAGTTTTT 4 ACP5_55540 54 ACP5 CGGTTTATTTTATAGATGCGGAGTTTATAGTCGGGAGGCGTGGCGGTTTT GGGCGGTGTAGGGTTTCGGGGCGGGGAGGTCGAGGCGTTCGTTTTT
ACTN2_55534 ACTN2 GCGGCGCGGTTATTAAGTCGCGCGGTAGTTGTTCGTAGTCGGAGTTGGT GTTTCGTTCGAGATTTAGCGTTTAGGCGTGTCGT
ACTN2_55535 ACTN2 GTGTTTTGTTTGTTTTCGGACGTGTTTTTTTTTTTAAGGGGTTTTCGGGGT
TGCGAGGGTATAGGATAGTATAGTATTTTTATAAAGCGTTTTTTCGTTAGT
CGTTAGGTTACGCGGTTA
ADAM23_2 8745 ADAM23 GAAGGACGAGAAGTAGGCGGTAGGGCGGCGTGCGGGTCGGGGCGTTG
GTAGGTATCGAGTCGGCGGGGTCGCGTTGGGGGTCGTAGGAAGCGTCG
GTAAGGTTGTAGTTCGTTAG
ADAM23_A 8745 ADAM23 GAGGTTTTAAGTTGGCGGAGCGGCGAGGATTTTTGGATTTTTTTGCGTTT
CGTTTCGGGAGTGGTTGCGAGGTTAGGCGAGTCGGGAAAGGGGGCGTC
GTTTAGTTTCGAGT
ADAMTS15_56144 170689 ADAMTS 15 TAGTAGAAGTATGGCGTCGGGTAGCGTATCGTCGCGTTGTGGGAAGGG
GTTGGGTCGGGTTTTTCGGTCGTTGTCGCGGTTTTCGTGTAGTTCGTATT
TTGGA
10 ADAMTS15_56145 170689 ADAMTS15 GAGTTTTTGATTTTCGGAGTTTTCGGGATTTCGTTAGTAGTAGCGGCGGT
CGTTAATGTTCGGGTTTAGTCGGATGGGAACGATTATT ADAMTS18_56138 170692 ADAMTS18 CGTTTTAGTTTCGTTAGGTTTTTCGGCGGGTTCGAATTCGTAGTCGGGAA GGTATACGCGAGTAGGAGGGCGTATTTTAT
12 ADAMTS18 56141 170692 ADAMTS18 GGTGTCGTAAATTGTAGTTCGGTAGGTTCGCGTCGGGAGAAGGGAGGG
CGCGTAGGCGGTCGGAGGAGGGGAGGTTTTAGCGCGTTTTCGTTGCGA
TGTGAACG
13 ADRA2A_57683 150 ADRA2A GGTAGAGTTCGCGTTTTAGTTTCGGGTCGGGTCGGGTTAGAATCGTAGC
GTTTGGGGGAAGTTAGAGAGTCGGTAATCGTTTCGGGGATGTAAGGCGA
TAGATATAGGATTTTCGA
14 ADRA2A_57684 150 ADRA2A GTTTTTGTTTTAATTCGCGTTGTCGTCGGATTTCGGTTTATTTAGTAGCGT
TCGGCGTTTATTAGGCGGACGTTTAGGAGAAT
15 APC(2) 324 APC TTATATGTCGGTTACGTGCGTTTATATTTAGTTAATCGGCGGGTTTTCGAC GGGAATGGGGAGCGTTTTGGTTC
16 APC2 10297 APC2 GTCGTTTGTTTAGGTTCGGATCGGGTTTTGTTCGTTTCGGAGTTTTTGTT CGCGTCGCGGAGATTTCGGAGTTCGCGCGTTTCGAGGTTATTTCGGGTC
17 APC2_56103 10297 APC2 TCGGATGGTGAAGTTCGTGAGTGGGTGTGTGCGTAGGATCGGTTGTAGA AACGTTGACGTTTAGTTTATCGGGATTTAGTTT
18 AQP5_56090 362 AQP5 TCGGGATCGAGTTTCGTTTTTTAGGGAGTTCGGGGCGTACGGTATCGAG GAGAGCGCGGGAGTTAATTTGGGCGTATTATGCGTAGGG
19 AQP5_56093 362 AQP5 AAGAAAGTTCGGAGTAGCGAGATTTGGTTGTTTATTAAGAGGGTTAGGGT GATGGCGGGGTTGATGTGGTCGTCGTTTACGGGTTTTAGGGT
20 ARMCX2_13030 9823 ARMCX2 TTTTACGTGATTAGGAGTTGACGTGGGTAAAGGTATTTAAAGTTTTGATC
GTCGTTTTTCGTTTTTTTTTGGGTATTAATTTTATTGCGTAGTTCGTTGTCG
TGCGTTTAAT
21 ARMCX2_13032 9823 ARMCX2 TGGCGATGTAGTTTTTACGTGATTAGGAGTTGACGTGGGTAAAGGTATTT
AAAGTTTTGATCGTCGTTTTTCGTTTTTTTTTGGGTATTAATTTTATTGCGT
AGTTCGTTGTCGTGCGTTTAAT
22 ATP2A2 23347 488 ATP2A2 TTTTTAATTCGTTTTGCGGAGTTCGGGGTCGCGCGAGGGGCGGTTGTTT
GGGGGAGGGGGCGCGGGGTGATTTAGCGTTCGGCGAGGCGGAAGCGG
TCGTAAGAGGAGG ATP2A2_23355 488 ATP2A: GAGGGTTCGAGAAGCGAAGAGGTTTAGGGAAGGCGAGGCGAGGATCGT
AGTTTTTTTTTTTTTCGCGGGGTCGGCGCGTTGGTTCGATTTTTCGTTCG
TAGTTGTCGG
ATXN1_56027 6310 ATXN 1 TACGGGGAT AGATTTGGGAGCGTTGGGCGGGGAGTAGTTTAGTTTTGTG
GATAATGTTTTGTGTAAATTTATTTTTTGTTGTAAATTAAGTTTTATCGAGG
TTTTTGTCGGTTTAT
ATXN1_56031 6310 ATXN 1 AATTAAGAGCGGAGTAACGAATGTTTGTTTTTTAAGAAGCGCGAGATTTT
CGTTATTAGTCGGTTTTTCGAGGAGAAGGT
ATXN1_56032 6310 ATXN 1 GGAGTAGTTGGTTGTCGTCGTCGTAGTTTAAGGATTCGATTTTATGGGGG TGGGGGGTATTTTTTGGCGGGCGGTTCGGCGAGTCGTATAGAGTACGG
AXIN2_56020 8313 AXIN2 GAGAGATAGAGAGATTACGTCGATTGTTGAGAGGAATTGGAAGAAGAAA
AATTTTTAGATTTAGTGGGAAGAGTTTTTTTATTATGAGTAGCGTTATGTT
GGTGATTTGTTTTTC
BACH1_56015 571 BACH1 TTTTGTAATTTTTCGCGTGGGTTTTCGGTCGCGGCGATTTTTGTTTCGCG
TTATTGCGCGCGTTCGTTCGTTGATAGAGGTTTC
BACH1 56017 571 BACH1 TTTTGTGGGGTTAGCGTTCGTTTTTTTTTTTGTTGTTCGCGGGTATTCGG GTACGCGGCGATTCGGTTGAATTAGGGCGTTT
BEX1 55859 BEX1 GAGTATTAGTTAATTGGTCGTCGGTTCGTGGGGGTTGGTGAGAAGGAGG GTGAGTTTGGCGGTGACGTACGGTTTTTACGTGATCG
BEX1_12842 55859 BEX1 TCGGGGTTTTTATTTGGTTCGTTTTTTTTCGGGTCGGATGTTAGTTCGTC GAGCGTAGGGTAGCGGGGAGTTGGTAGCGAGATACGAGTGACGATT
BEX1_12850 55859 BEX1 TTAGTT AATTGGTCGTCGGTTCGTGGGGGTTGGTGAGAAGGAGGGTGAG TTTGGCGGTGACGTACGGTTTTTACGTGATCGGGAGTTGTAGAGT
BHMT2_55998 23743 BHMT2 TTCGTTTAAGTGTTTTGTCGAGGTTTTAGGGTTTGGTTAGGGATGGACGT CGGGTGTGAACGGAGTCGTTCGTAGTTTAGCGGTT
BHMT2_56001 23743 BHMT2 TTAAAGAGTTGCGGGCGAGGGTCGGTTGAGGGAAGAAAATGTGTGCGC
GATATGCGTTCGAAAGTTAGATTTTTGATTTTCGTTTTTCGCGGGGTTGG
ATTCGTGATTTTGAGTTAGTCG
BIK_17885 638 BIK TTTAGAGTTCGGAGTCGGGTGTTCGGAAGTCGTATTGGAGGATTGTGCG
GCGGCGATGGGGCGTTTAGTCGTCGCGGCGAGACGTAGAGGGATTAT
BIK_22360 638 BIK TTTTTGGAGTTTCGGTTTTTACGTGGGTAAAATGATCGTGAAAAAAAGTAT CGAGGAGTATTTTGAGTTCGAACGGAGGTTATTCGTG
BRCA1_JH 672 BRCA1 TCGTGGTAACGGAAAAGCGCGGGAATTATAGATAAATTAAAATTGCGATT GCGCGGCGTGAGTTCGTTGAGATTT
C10orf13_11683 143282 C10orf13 CGTAGGTGTATGTGTTTGGGCGAAGCGTTGCGTGTGGTTTAGTTGGTGG
TAGTTTTTGTTTTTGTTCGTAGGTTTTGGGGAATGTTTTTTGTTTGGTTTA
GTTTCGATAAACGTGGTTTGAGA
CALCA_2 796 CALCA CGTTTTTATAGGGTTTTGGTTGGACGTCGTCGTCGTCGTTGTTATCGTTT
TTGATTTAAGTTATTTTTCGTTAGGTGAGTTTCGAGATTT
CCK 885 CCK TTAGCGTTGGGTAAATATATGATTGGTCGACGTCGTCGGGTGGGGTTATT
TAAGAGATAGTCGTTCGTTGGTTTTTTTTGAATTTGGTTTAGTTGTCGGGT
TGTTTCGGTTGGAAAC
CCK_13381 885 CCK TTAGTTGTCGGGTTGTTTCGGTTGGAAACGTTAAGTTAGTTGCGTTTTAAT
TTAAAAGGTAGGTTTTTCGGTTCGTTAGCGGGGGATTTAAGTGTC
CCK_13382 885 CCK ATTGCGAGGGTTTTTAATGCGGTTGAGAAGAAAGTGAAGATTTCGATTTT TTTTTTTTTTTTCGAAAGAGTCGATAT AGGTATGAAGATTAGCG
CCND2_1 894 CCND2 GAGTTTCGGGGTTGTTTTATTCGTATCGGTTTTTTTTTTAAAATTGGTTTC GTTTTTTTTTGTTCGTTTTTTTTCGTTTTGAAGCGGTGACGTAAGTTGG
CCND2_25209 894 CCN D2 GAAGGTAGCGTTTTTCGATGGTGAGTAGGTTTTGTAGGACGCGGTCGTT TCGGAGTAGGTTGCGGTTTCGTACGGTTTTGCGGATCGGGTTTATTT
CD34_55940 947 CD34 GCGGTATTTTGGGTTTTGCGCGCGCGTTTTTGCGGATTAGTATTTTTTTC GCGCGGTTTTTAGAGAGACGTATCGAGTGGAAGATATTA
CDKN1A_24717 1026 CDKN1A GGGAAATGTGTTTAGCGTATTAACGTAGGCGAGGGATTGGGGGAGGAG GGAAGTGTTTTTTTGTAGTACGCGAGGTTTCGGGATCGGTTGGTTT
CDKN2A_8472 1029 CDKN2A GTTTTGGCGAGGGTTGTTTTCGGTTGGTGTTTTCGGGGGAGATTT AATTT GGGGCGATTTTAGGGGTGTTATATTCGTTAAGTGTTCGGAGTTAATAGT
CDKN2A_8475 1029 CDKN2A GTTTTCGTGGTTTATATTTCGCGGTTTACGGGGGAGTGGGTAGCGTTAG
GGGCGTTCGTCGTTGTGGTTTTCGTGTTGATGTTATTGAGGAGTTAGCGT
TTAGGGTAGTAGTCGTTTT
CDKN2A_9696 1029 CDKN2A TTTAAGTTTTTAGGGCGTCGTTAGGAGGAGGTTTGTGATTATAAATTTTTT
TTGAAAATTTTTTAGGAAGTTTTTTTTTTTTTCGGAGAATCGAAGCGTTATT
T
CDKN2A_9700 1029 CDKN2A CGGTTGTTGTTTTAGACGTTGGTTTTTTAGTAGTATTAGTACGAGGGTTAT
AGCGGCGGGCGTTTTTGGCGTTGTTTATTTTTTCGTGAGTCGCGGGATG
TGAATTACGAAAAT
P16 NEW1 1029 CDKN2A GAGGGTGGGGCGGATCGCGTGCGTTCGGCGGTTGCGGAGAGGGGGAG
AGTAGGTAGCGGGCGGCGGGGAGTAGTATGGAGTCGGCGGCGGGGAG
TAGTATGGAGTTTTCGGTTGATTGGTTGGTTACGGTCGCGGTTCGGGGT CDKN2B_27345 1030 CDKN2B TTAGAAGTAATTTAGGCGCGTTCGTTGGTTTTTGAGCGTTAGGAAAAGTT CGGAGTTAACGATCGGTCGTTCGGTTATTGTACGGGGTTT
CDKN2B_27347 1030 CDKN2B TTTTTATTTTGTTAGAGCGAGGCGGGGTAGTGAGGATTTCGCGACGCGT TCGTATTTTGCGGTTAGAGCGGTTTTGAGTTCGGT
CDO1_55928 1036 CDO1 AATTTGATTTGTGTGTGTATCGCGTTTTTAGCGATTTCGGATTTATTGCGT
TGTTAGGGGTTTGGGGGTGGGTTTTTTGTTGTTTTTGCGACGATATTTTT
ACGTTTC
CDO1_55929 1036 CD01 GTTTACGCGATTTTTGGGACGTCGGAGATAACGGGGTTTTTGGGAAGGC
GCGGAGTTCGGGGAAGTCGGGGATGTGCGCGTGAGTCGTGTTCGTAGG
GTTTTT
CFTR_55912 1080 CFTR TCGAGAGATTATGTAGAGGTCGTTTTTGGAAAAGGTTAGCGTTGTTTTTA
AATTTTTTTTTAGGTGAGAAGGTGGTTAATCGAGTTTCGGAAAGATACGT
GTTTACGAAAGAGGAGG
CFTR_55913 1080 CFTR AGTTTTTCGGGGAGTCGGTTTTTTCGTCGGTGGTTTTTTTTGTTTTTTAGC
GTTGTTAATTGGATTTAAAGAGAGGTCGCGATTGTCGTTTATTTGC
CHL1_55888 10752 CHL1 GAATCGAGTGAAATTATCGGGGAGGGGGTGGGGGGCGTTTTTTTTAAAT
GTCGTTTTTGTAGATAAACGAGTAGGGATTTTATTTTTTTTGATCGGAGAT
GTTGAAAAGTACGATT
CHL1_55889 10752 CHL1 ATTTTGATCGCGTTTCGTTTTTTTTTTATAACGAGTGGATTTTTTTCGGAGT
ATTTTTAGTTTTTTTTTTACGGGAGATTTTTTTAGCGTCGGTACGAGAGAT
GTTAGCGGATTTT
CKM_55887 1158 CKM AGT AAACGTAAAGAAAGTTTGCGAT ATTTGTTTTTGTAGATTTTGTTAGGC
GATAGATATATTTTTTACGGTTTGTGGCGTTAGT
CNIH3_55877 1491 11 CNIH3 AAGAGTTTTTGTTTTAATCGGTTTCGTTTTATTTTCGTTCGGTTTTTAGTTT
TGTTTGTATTGTTGGTTTTAAGTTTTTTTCGAGAGGCGGGCGTGTATAGTT
GTA
CNIH3_55879 1491 11 CNIH3 TTATTAAATCGTTGAGGAGAGACGAGCGTTTCGTTTCGGATTTTTTCGCG
TTATCGGGGCGTAAACGGATAGTGTTTAGC
CNN1 55871 1264 CNN1 GTTGATAGTTCGTAGGTAGGGTTTCGGTTGAAGTGAGTAGAGGATATGTT GGTCGGTGGGTTTTGGCGGGGGTAGTGGCGGTATTGACGTTGAGAATA
CNN3 1266 CNN3 GTTTTCGCGGTTTTTTAATTGGTCGTATCGTTTTTCGGCGTAGAGGCGGG
AGGAGGCGGTTTTTTATTGGTCGGGTTCGGCGGTTAATTTTCGAAGGTTT
AGGC
CNN3_13362 1266 CNN3 TTTAGGTATCGTTCGCGTTTCGCGGTCGTTTTTTGCGTTTTTTAGAGGAG
GGAGTTCGAGTATTTTTCGCGGCGGTTATTTTTCGGGGTTAGGAGCGGG
GAAGCGAAGTGCGAGAGATTT
COL7A1_55842 1294 COL7A1 CGGTTATTAGAAGTCGTAGCGTTATTTTAGGTAGTAAAAGTCGTTAGTTA
GGATTTTCGTTTTTGTTTTTTGTTTTTTCGTGGTTTTGGTAGGTTC
COL7A1_55846 1294 COL7A1 GGGTGGTACGGTGTAGTGTTTCGGGTCGGGTTTTTTTTTGCGGTGTTTAT TTTTTTTTTTGTGTTGGGTTCGTATTCGGGGCGTTTTTGTTA
COL9A3_161 15 1299 COL9A3 TAGGTTTACGGGGGTATTTGCGTTTTTTAATGAGTTTTTTTCGTTTTAGAG
AGTGGGATTTTTCGGTTTTTTCGGTTTTTTAGGGTCGTTCGGGAAGTTCG
GTTAGGA
COL9A3_161 18 1299 COL9A3 TCGCGTAGTGTGTTTTTCGTTTTTTTTCGGCGGCGGGAATAAAGGTTTTT
TTGTGTTTTTAGAGGAGGCGGCGGCGTCGGTCGATTCGGGTTAGCGAG
GATTTAT
COL9A3_16187 1299 COL9A3 GGGTTTATTTCGAGGCGTTGGCGATTTCGCGTTTCGACGGCGATACGGG
TTGGGGGCGTCGAGGTTGCGTCGGGTATTTCGGAGTGGCGGGTTCGGG
GTTGGGGCGGTCGCGGTTTTACGTTTA
COX7A1_55819 1346 COX7A1 GTAGCGTGATAGTTGGGGACGCGGCGGGGTTGGGGTTGGGGTTGGGG
TTGGGTTGCGGGGAGTTTTGTTTTTCGGATTCGTTTATTACGGAGGAGGC
GGTTTAGATTCGTATTTT
CRIP1J 1396 CRIP1 GTCGTTTTAGGGATTTAGCGTTTTCGGTTTTTTTGAGCGGTTTTTAGTTTC
GTCGTTTCGTCGGCGGCGTTTTTTCGCGGGGTCGGTGTGGGCGCGTTTA
TCGAAGTATATT
CRIP1_2 1396 CRIP1 TTTCGTTTTAATTTTGTAGCGTATTTGGATCGTTTTGGTCGTTTTGGGCGT
TATTCGTAGAGATAAGGGTTTTTTTTTGCGTTCGGTTCGGGTATTTTCGA
GT
CRIP1_4 1396 CRIP1 AGAGGAAACGGTAGAGATCGTAGGATTTAGAGACGGGGAGGTTTAGAGT
TAGAGGTAGTTCGTCGAGTATAGTAAGATTATTAGTAGGTAGAGATCGTT
AGGGTTTTCGGACGTAGGGGTTTTTAT
CSPG2_1 1462 CSPG2 TTAGAAAGGTTCGGGGTAGTTCGTTTGTAAGTTTAAATGCGGGTTGTGAT
ATTTATTATTATTATATTATTGTATCGTTAGAGTCGAGGAGGAGATT
CSPG2_23363 1462 CSPG2 TATTGTAGCGTTGCGCGATTGGGTTCGGCGTTGTTTAGGCGGGTTATATA
GGAAGCGTGGTGGTTCGGGGAAGGATGCGGAGGGTGCGGGACGGTGT
TGGAAGATGC
CST6_17991 1474 CST6 TTAGTTTTAGGTCGCGGGGGCGTATCGCGGGCGTCGGGCGGGGCGGTT
TAGCGGGTAAAAGTTGCGCGGTCGTAAGTTCGGTATTTACGGTTTTGAG
GGTTTCGACGGTATTGACGGT
CST6 17993 1474 CST6 GGGAAGCGTTTTGGTACGCGGGTGCGCGTCGTTTTTTCGGTTTTTTGGG
TTTTTTGAATTTCGTAGGATTTCGGTAATTTCGAGTTTCGTTT 79 CST6_55797 1474 CST6 ATGCGATTAGGGTTAGGTTTAGCGTTAGCGGGAGGTTCGAACGCGTTAT
GGTCGTTAGTGTCGTCGGAGTTTTTAGAGTC
80 CTAG2 246100 CTAG1A GTGAGAATCGGTTACGTGTTTCGGGGTTTATTCGGGGTTTTTTAGGGTCG
GAAGTAGGGTTTTTGTGCGTAGGCGTTTTGAGGATTTC
81 CXCL1_55776 2919 CXCL1 GAATTTCGTGAGGTAGGAGGTCGGGTTGGGGATAGGGTTGGGGTAGTA
GGAGGGTTTGGGATTTCGGCGGTGGTAGAGGTAGCGCGGGGCGGGATT
TATATGATTTC
82 CYCLI N D2_2 894 CCN D2 GGTGTAGCGTTTAGGGTCGTCGTAGGTCGGGGGTAGGGTTTTTAGCGGT
TTTTTCGCGGTTAGCGGTTACGTAGGAAAAATTCG
83 CYP1A1_23831 1543 CYP 1A1 TATTCGTGGTGGTGTTGAGCGGTTTGGATATTATTCGGTAGGTTTTGGTG CGGTAGGGCGATGATTTTAAGGGTCGGTTCGATTTTTATATTTT
84 CYP24A1_55769 1591 CYP24A1 AATTACGGTCGTCGTTGTCGGTTTTTGTTCGTCGGGGGAGGGCGGGGA GGCGCGTTCGAAGTATATTCGGTGAATTTCGGGTTTCGTATGATTTTT
85 CYP24A1_bay 1591 CYP24A1 TTATTTTAGGTTCGGACGTTTTCGTTTATTTCGTTGATTTTATTTTTTTTTTA TTTTTTTTTTTTTGGGTTTCGCGTTTTTCGGAGTTTGGTTAGTC
86 CYP26A1_55745 1592 CYP26A1 CGTGGAAGAGAGTTTATTCGGTATTTGGAAATGGAAAGTTAGTGAAGGTT
GTTTTGGGTCGGGGT AGCGGGTGGGATCGGGCGGGAGGGATTTTAAAG
AGATCGTCGGGAAGGTTAGAGT
87 CYP26A1_55746 1592 CYP26A1 GATTTGTTTATTGCGTT CGATGTTTCGAGGTTTTTTTTTGGATTTTGGTTTT
GAGTTTTTTTGCGCGATTTTTCGGAGACGTTTGGAGGTTT
88 CYP26A1_55754 1592 CYP26A1 AGAGCGTATTGGTTAGTAGCGTCGGGAGTTTTATGGCGCGTCGCGATTT
TTCGCGTTATTTGTCGTCGTCGTTAGCGTTTTAAATTTTACGGCGTTGTC
GTTTTATAG
89 DACT1_23462 51339 DACT1 GTCGGGATAGTAGTAGTCGGCGGTCGCGCGTAGGATTCGAGGGTTTTTA
GTTATCGTTTTCGTTAGCGTCGCGTTTCGTTATAGGGCGGTATGA
90 DAPK1 1612 DAPK1 GGATAGTCGGATCGAGTTAACGTCGGGGATTTTGTTTTTTTCGCGGAGG
GGATTCGGTAATTCGTAGCGGTAGGGTTTGGGGTCGGCGTTTGGGAGG
G
91 DDX27_55724 55661 DDX27 GTTATCGTTTTGAGGTCGGAGAT AATGGAATTTGTTTATTTAAAAAGTTTC
GTTTTTTGATGGCGTTATACGAAATTAATTACGTTTTATATTGTCGTATGC
GTATTTTTAAGAG
92 DDX27 55728 55661 DDX27 TTTTCGGAGTTAGATTCGGGTTTTATCGGTATTTCGTTATTTTCGTTTATG
GTTTCGATTAAGTCGAGGTTCGTAAGTATGTTGTCGTAGAAG
93 DDX43_8890 55510 DDX43 GTGTAGAGTTGGACGGTAACGACGTCGGACGCGTTTTTTTTTGGAATAAT
GTTTTATTACGGAGGAGTTTTTAAGGTTTTTACGTGGGTCGTTGTTAGTC GGCGAAGTT
94 DDX43_8891 55510 DDX43 TTGCGTGAAATTAGTGCGAAGTGGGCGGGATAGAGAGCGTGGGCGGGG
GGGTTAGTTTCGTGCGGGTTTTTTAAGTAGCGGTTGCGTGGTTTTTTTGG TACGTTATTTTTACGACGTTACGG
95 DNAJA4_8894 55466 DNAJA4 TTTTTAGTTTTATTTTTCGGCGTAGGGTTTCGGTTAATATAGTTTTTTAGGT
CGTTTATTTTTTAGTTAGTCGGTTTTACGGATTTACGGAAGGGTAAG
96 DNAJA4_8897 55466 DNAJA4 TTTCGTAAGGAGACGGTGACGTTATAAAGGTTTAGAATTTTCGCGGAGGT
TTCGGCGGTTTCGCGGCGGGTTCGCGGTTTAGATTTTCGAC
97 DSC3_52537 1825 DSC3 TTTACGTTTTTCGGTTGTTTCGCGGCGTTGTCGTTTCGTTCGGTTCGTAG
AGAGGTTTTGGTAGTTTAGATCGTTTTAGGGCGGTTTTATCGTCGGCGTT GCGCGGTGTATTTTAATGTA
98 DSC3_52538 1825 DSC3 TTTTTGGTTTTGTTCGGTATTTCGATGGTCGTCGTTGGGTTTCGGCGTTT
CGTGCGCGGAGTCGTTTGTTTGTATTTGTTGTTGATTTTCGTGGTAAGTT AGATTTCGTCG
99 DSC3_52554 1825 DSC3 GTCGGGTAGGGTTAGGAGAACGCGGGCGTCGGGAGGGTGTCGAGAGC
GAGATTTGTCGAGGTGTAGGGCGCGGGAGGTGTTTTTTTCGTCGTTGTT TGTTTTTAAG
100 DUSP6_18075 1848 DUSP6 CGTATTTATTATGGGGGTCGAGTTGCGGGAGAGGGCGGGGTGTTTATTA
GACGTTTTTCGGGGTAGGTATAGGTCGAGCGTATCGCGCGCGAAGTTGT CGTTTTCGGAGCGGGGTTTAAT
101 DUSP6_18077 1848 DUSP6 AATTTGTTTTAGTCGGTTCGTTTTTACGGTAATAGTTTTTTTCGTAGTACG
TTTATTGGTTGGTTCGGAGAATGTATTTATTGAGACGT
102 EIF5B_55689 9669 EIF5B GGTTGTTGTTTGGTCGGTCGGTTAATCGGAGACGTTTTATTCGAAGCGTA
GTTTTCGATGAGTCGCGTTTTGCGTTTTTACGT
103 EIF5B_55693 9669 EIF5B GGTTTAGGTTTGGTATTTCGCGAAGTTTATCGCGAGTTTAGATTTCGGTT
CGACGTCGCGTTCGGCGAGATTCGTATATTTTATTGAGTTACGGCG
104 EPB41L3_19071 23136 EPB41L3 GGGATAGTGGGGTTGACGCGTGGTTTCGGCGTCGCGCGGTTTTTCGAAT
TTCGAGTTTCGCGTTCGGCGCGGTCGGGGTTTTTAATCGTTTTTTCGTTC GTCGGGATTTTTAT
105 EPB41L3_19072 23136 EPB41L3 GCGTGGGTTTTCGTCGTAGTTTCGCGGAGTTTCGGTGTTTTTTGTAATAG
GGGGCGGGGGGAATAGCGGCGAGTAGTTTTGGG
106 ERCC3_2A 2071 ERCC3 GGATTTTCGTCGTTGGGTCGTACGAGTTAATAGATCGGCGGTCGTTGGT
TTTTCGTTTTTTTAGTTATTTATATTACGGCGTTTAGAGGCGGGATATTTT 107 ERCC3_2B 2071 ERCC3 TTATGGGTAAAAGAGATCGAGCGGATCGCGGTGAGACGTTGCGCGGGT ACGTTTAGTTACGATTGTTTTTGTCGGTTTTGTTTTTCGTTT
108 EREG_18102 2069 EREG TTTTTGGTTTTCGGTCGTTTTAAAGAGGAGATAAATAAGGAAGGTTTTTGT GTATTTGTTATTCGATAAGTACGGAGTTGTTTT
109 EREG_18104 2069 EREG TTTTCGGGTTTTAACGGGGTGAGGTTAAGAGTGTTTAGAGTTTTATAATT GGTTCGAGGGAGGAGCGGTAGGAGGATCGTTA
110 EREG_18105 2069 EREG TAGCGTAGGGATTTTGTCGGTATAGAGTATTTTTATTTTTTTTTTCGCGGT
TATCGGCGATGGGAGCGGGCGTTGGGGTTTGGCGGAGGGCGGTTGCG
GAGAGTGTACGGGTAG
111 ESR1_2 2099 ESR1 CGGGAGTTTAGGAGTTGGCGGAGGGCGTTCGTTTTGGGATTGTATTTGT
TTTCGTCGGGTCGTTCGGTTTTATCGGATTCGTAGGTTTTCGGGGTAG
112 ESR1_24372 2099 ESR1 GGTAGGGTGTAGATCGTGTTTTCGTAGGGTAGAAGGTTTAGAAATCGGC GGGTTATTTGGAAAAAGAGTATAGTTCGAGGTTAGAGGCGAC
113 ESR2_55651 2100 ESR2 GATTGTAATTTGAGCGCGGTTTTTTTAGTAGAGTAAGTATATTTATCGGAT
TTAGTAATTTAAGGGGTTAGTTTTTTTTAGTATTTTAGTTGTCGTCGTTTTG
TTAGGTTTT
114 ESR2_55653 2100 ESR2 TTTTTAGTTTTGGGGACGCGGTGTAGAAGTGTGAGGGCGTTCGGTTTTTA
GGTAGTAATGGGCGGGTTTTTGCGCGGGAGCGTGGCGGGCGTTGGATT
TTATAG
115 ESR2_55656 2100 ESR2 TGAGTTGTAGGAGGTGCGTTCGTTTTTTTTAATAGGTGGCGGCGGGGCG
CGCGTCGGGAGATTTTTTTTAATGCGGGAAAAGTACGTGTT
116 ESR2_55657 2100 ESR2 TTTTCGTTAGGAGGTAGTTGTAAGCGCGGAGGTTGCGAGAAATAATTGTT
TTTTGAAATTTGTAGGGCGAAGAGTAGGCGGCGAGCGTTGGGTCGGGG
AGGGATTATTC
117 EYA4_22346 2070 EYA4 TTAGTTTGGAGGTTGTAGTCGTTCGAGTCGGTTCGGGTGGGGGCGGGG
TGGGGGCGGCGCGGAGGGTACGGAGATTACGGCGGCGTTATTCGGGAT
ATTTAGGG
118 EYA4_22350 2070 EYA4 GAGAGAGCGCGTTTAGTCGTCGTAGTCGTTATTTTTCGGTTTTTTTCGTA
GTTTTTTTTTTTTTTTTAAGGTAGCGATAATTTTACGTGGATAGGA
119 FAM20B_55630 9917 FAM20B ATATAGGGATACGCGGTTCGAGCGGGTTTTTTAGGAAGTTTCGGGTTTTC GTTCGGGATTCGTTCGTAGTTATTCGGGGACGG
120 FAM84A_55594 151354 FAM84A CGTAGATTTTCGTTTTTGGTTTTCGTTTCGTTATTTTATTGATGGGTAATTA ATTGGATCGTATTATTTATTTTAATTATAGCGAGTTGTTTATAGGGGATTC
121 FAM84A_55600 151354 FAM84A GTCGGTTAGTTTACGTGTTTTTATCGGAGTTATTTTTTTTAGTCGAGTTTTT CGTGTTTTTTTAGGGGTACGAGGGGCGGAGAAGTCGTTATAG
122 FAM84A_55616 151354 FAM84A GTTTTGTTTTGTTTCGTTTCGTTTCGTTTCGTATTTCGTATATTCGATCGC GCGTCGTTTCGGGGAGCGGAGGTGTGCGTATT
123 FAM84A_55624 151354 FAM84A CGGAGTAGTCGGTTTTAGGGTTCGTTTTAGGACGCGTTTATTGGTTGGG GCGGGTGTTCGAGTTGTGTTAATTTTCGTCG
124 FAS_18137 355 FAS TTAGCGTTTTGAGTATTCGGGTTTGAGGGAAGGTTTTAATAGTTTTCGGG
AGTTAGTTTTTAACGTTTTTCGTAGTAGTTCGTCGTTTTTAGGTGTTCGCG
TGCGTCGTTGTCGTCGTA
125 FAS_18139 355 FAS GGAGTTGTTTCGTTTGTTTAGCGGGGTTAGTATTTCGTATTAAGGTTTAA
GAAAAGTAAGTTTTTTAGCGTTTTGAGTATTCGGGTT
126 FAS_18143 355 FAS TTTTTGATTATCGGGGTTTTTCGTGAGTTCGTTTTTGATTTCGCGTAAGAG
TGATATATAGGTGTTTAAAGACGTTTTTGGGGAGTGAGGGAAGCGGTTTA
CGAGTGATTTGGTT
127 FAS_18147 355 FAS GTGCGTCGTTGTCGTCGTAATTTCGTACGCGTTTCGCGTTCGTTTTATTT
TGTTTATTTTCGGGATTAAGACGGGGTAAG
128 FBLN1_54935 2192 FBLN 1 ATTTCGAGTTTTCGTGGTTTCGAAATTCGTAGGGAGCGCGAGGTCGAGG
GGGTCGGGATTTTAAGTTGGGGCGGCGGATTCGGGACGTTTTTATTTTC
GGTCGTTAGTAGCGTAAGGTCG
129 FBLN1_54936 2192 FBLN 1 AGATGAGGATCGGAGTTGGTCGTGGGGGAGGGGGGCGTCGTCGAGGTT
TCGGCGTTGATAGTTCGGGGGCGTCGCGTAGGGTAACGTACGGATTTC
GAGTTTTCGTGGTTTCG
130 FBLN2 2199 FBLN2 TTCGTCGGAGAGGGGGTCGGGTCGGCGTCGTTCGTTTAGAGTTTAGATT
CGTTGATTCGGTTTTTAGAGGTCGTT
131 FBLN2_13328 2199 FBLN2 TAGAGCGGAGGAAGTTGCGGATTTGGGGTGGGGGAATTCGTTCGCGGA TTTTTGGTTTTTATTTCGCGTCGGTTTTTGTGTTCGTATTTG
132 FBN2_18150 2201 FBN2 TCGGAGTTTTATAGGGTAACGAAGCGCGGGTAGCGGTTGCGGAGTCGG GCGGAGGTGCGCGGGGTCGGGGCGTGCGGTTAGTAAGAG
133 FBN2_18151 2201 FBN2 TTGGAGATTTCGATAGAGCGTCGGTTTTTTGATTGTTCGCGAAGCGAGAC
GCGGGGCGTCGGGTTTAGCGTAGTGAGCGGCGAGGCGCGGCGGAGGT
GTAGTCGGTAGTTT
134 FKBP4_31086 2288 FKBP4 TTCGTAGGTAGCGTTTTCGTTCGCGGTTTAGAGTGCGTTCGCGTCGGTA
TTAGTTTTCGGATAAACGGCGCGTCGCGCGGAGATGATAGTCGAGGAGA
T
135 FKBP4 31088 2288 FKBP4 TATTAGTTCGTTGCGTTTGCGGCGGGGTGTGGGGAGCGTAGGTTGCGG
GATGGTAGTGGCGGCGTTTTTTTTTCGGGATCGGTCGTTATTAC 136 FLJ21511_57795 80157 FLJ21511 TTGTATTTTCGGGGTTGTTTCGGGTTCGGGTTTAATAGTAGAGTTAGAGT
CGAGACGTATGTTTTTTGTATCGGTTAAGGGTGTCGTTGGGGTTCGCGTT
AAGGAGGTAAAG
137 FLJ21511_57802 80157 FLJ2151 1 TATTTTTATTTTGGGTACGGAGCGTTTTTTTTTTTTTATTTGAATTTAGTTA
ATTTGGTTTGGTTTTACGGTTTTGGATATGGTTCGGGGAGTTGGTATTCG
138 FOS_22333 2353 FOS GCGTAGGTAAGGTTGGTTTTTCGTCGTCGCGGGGTCGGGGGTTTGGGG TCGCGGAGGAGGAGATATCGGGCGGGACGTTTTAGTAG
139 FOS_22338 2353 FOS CGGGTTGTAGTTAATATCGAGGGTGTAGTGCGGGGGGAGGCGGGGGTC GCGGTTGGGGGAGGGGAGGCGGGAACGGCGTAGAATGAGAGAG
140 FOXL2 668 FOXL2 GCGATAGGTTTTTAGTAAGTAAGCGCGGGCGGTATTCGTAGTTTTTAGAA
GTTTGAGATTTGGTCGTAAGCGGATTCGTGCGTTTTAATTTTTTGTCGCG
TTAGCGTTTGGAGCGGAGAG
141 FOXL2_13395 668 FOXL2 GTTTCGTTATAGGGGCGAAGGCGTTTGACGTAAGCGGAATTCGGTGGAG
TTTATACGAATTAGAATAGAGCGAGGTTTTTGGCGTATTAG
142 GADD45G_57135 10912 GADD45G TATATTAGAAAGCGGGTGTCGGTTAATAGGCGCGTAGTTTCGCGTTAGTT
GGCGGCGTCGTATTTATTAGTTTATATAAGGGCGCGTAGCGTAGTAGGG
GCGTATTC
143 GADD45G 57137 10912 GADD45G GTGAGGTAGTTTTGACGTTGCGTCGATAGTAGTAATTTATGTAGCGTTTT
TTCGGTATTTTGTATTTTGCGGAGGAGTTAGCGTAGTGAGCGGGAGTC
144 GALE_57121 2582 GALE GAAGTACGAAAGTTGGTTTGAGGTCGGTTGCGTTTTCGATAGCGGAGTG ATTTTGTTTAGTCGTCGTTTATTCGGTTTTT
145 GALE_57124 2582 GALE ATTCGAGTAGGTTCGGGAGACGGAATGAAGGGTCGGGTGGGTTCGCGA
GGGGTCGGGGAGTTCGCGGGGAGCGAGGGAGTTTTCGAGGGGCGGGT
CGTGTTTTTGGTAGCG
146 GALE_new2 2582 GALE GTTATTGGGATTTGGCGTCGGCGGCGTTTTTCGCGGGGTGGTATCGTTT
ATTTTCGTTTTTCGCGGGCGCGCGTTGCGTAGTTTTCGTTATAGC
147 GDA_57106 9615 GDA TTCGATTAGTAGATTCGCGTTGCGTTTCGTCGTTGATATGTGTGTCGTTT AGATGTCGTTTTTGGCGTATATTTTTCGAGGGA
148 GDA_57108 9615 GDA GTATCGGTAATCGTTCGGGTAAGCGGGGGTAGGATAAGGTCGGAGTTTG TGTTCGTTCGGTAGTCGTTCGTAGTTGTAGAGAGTTTCGTTG
149 GDF10_57718 2662 GDF10 CGGGGATATGAGTTATGGCGTGGTGAGGGCGGTAAAGGGTCGAAGTTT AGGAGGAGGAAGGCGAGCGTTGGCGTATCGGAGGTTGCGGATTG
150 GDF10_57726 2662 GDF10 GTTATAGTCGTTCGGAGTAGCGTAGAGTCGAGTCGAGTTCGAGTCGGCG CGTTGTTTTGGCGGATTCGCGTCGCGAAAGTTTGTAGTTTATTGCG
151 GDF10_57728 2662 GDF10 TAGTTGGGGTTCGGGTTTCGGGTTGGTTCGAGCGGGGATATGAGTTATG GCGTGGTGAGGGCGGTAAAGGGTCGAAGTTTAG
152 GDF10_57734 2662 GDF10 TTTTTATATTCGCGGGCGTATATTTCGGCGCGCGTACGTTGTTATATACG GGCGTACGTATACGGTAGTCGGGTTAGGGACGATTTT
153 GJB2_22303 2706 GJB2 GTATTTCGGGCGGTGTTATCGCGTTTATTTTTTCGGTCGTTTTATTTTAGT
TTCGGAGTTCGGTCGTAGAAACGTTCGTTTTAGAAGGCGGTTTTCGTTTT
TCGGTTTAAGGACGTGTGT
154 GJB2_22306 2706 GJ B2 TGCGGAGTATAGAGGATAACGATTATAGTTATTTTTGAATTTCGTTTACGG
TATAGCGTCGGAGTCGGGGTTTGGGGCGTCGTTTTTTGGGGGGTTTCGA
TTTTTAGTCGTTTTCGTT
155 GPNMB_52603 10457 GPNMB TTAATTTTTAGTTTTTCGATTGCGATTGCGATTCGGGAAAACGTTTACGTT
TTTGGAAAGGAAGTTTTATTATAGGGAGTTGATTCGGTGTTAGCGCGTAG
TAC
156 GPNMB_52605 10457 GPNMB GAAGCGGTTAAAGGCGTAGCGGTTTTTGGTTAGAGTCGTAGAGGTTTGA
GACGTGGGTCGCGTTTCGGTTTAGGAGATTTCGCGGTCGCGTTTTAATTT
TTA
157 GPX1_57073 2876 GPX1 GTTTAAGAAGTTAGCGGAGCGTTTCGAATAAGTATTGTAAGGGGAGGTTA
GTAGGCGTTTTTTTTTAATTGGTCGGCGGCGGGTTACGTGGTATAGG
158 GPX1_57074 2876 GPX1 GGTAATTTGGATCGTTGTCGTTTGGTTGTTTGGGTTAGATTAGATATGTTT GTTGTTTTTTTCGGTTTAGGAGGAGTACGC
159 GPX7_57065 2882 GPX7 CGGGGTAAATTGGTGTCGTTGGAGAAGTATCGCGGATCGGTGAGTGCG
CGGGGTTTGGCGGCGTCGTTGGGTTCGGTTTCGTTTTGGCGGGGTTTGT
TGGGGACGTTTCGTAGTTCGGTT
160 GPX7_57070 2882 GPX7 CGGCGAAGGTTTGGATTTCGTTGTTTAAAGGTGTCGGTAGCGAAGTGGG
CGTCGATTGCGAGCGTTTCGGGTTTTTTCGCGTTAAGGTCGTGTTACGT
161 GREM1_29775 26585 GREM1 AGGTTTTGTATGTGACGGAGCGTAAATATTTGAAGCGAGATTGGTGTAAA ATTTAGTCGTTTAAGT AGATTATTTACGAGGAAGGTTGTAATAGTCGT
162 GREM1_29777 26585 GREM1 GAATTTGGTACGATTTTACGGAGATTTCGTTTTTTTTAGCGTAGTTTTCGT
TATTGAGCGCGGGATTAACGTAGGCGATGTCGGGCGGTCGATAGGGAA
AGTTTAGAT
163 Gst-Pi 2950 GSTP1 TTCGGGGTGTAGCGGTCGTCGGGGTTGGGGTCGGCGGGAGTTCGCGG
GATTTTTTAGAAGAGCGGTCGGCGTCGTGATTTAGTATTGGGGC
164 Gst-Pi New1 2950 GSTP1 TCGTTATTAGTGAGTACGCGCGGTTCGCGTTTTCGGGGATGGGGTTTAG AGTTTTTAGTATGGGGTTAATTCGTAGT ATTAGGTTCGGGTTTTCGGTAG GGTTTTTCGTTTATT 165 Gst-Pι_New3 2950 GSTP1 ATTTAGTATTGGGGCGGAGCGGGGCGGGATTATTTTTATAAGGTTCGGA
GGTCGCGAGGTTTTCGTTGGAGTTTCGTCGTCGTAGTTTTCGTTA
166 HCP5_57050 10866 HCP5 TTGTTTTCGGGAGTCGTGGTTTTGATTTAGATTTGGGTAGGTGAGTGCGG
GGTCGGGAGGGAAAAAGTTTTTGCGGGAAGGAGCGAGGGGTTCGTTCG GAGGAGTAAATT
167 HHIP_23319 64399 HHIP AGTAGTAGGAATAGAAACGGCGACGGCGGCGGCGGGGTAGGCGGAGG
TAGGGTTAGCGTTGGGTTTTAGATGATGTTGAGGTTTTTTTTGTCGGCGG
TTGTAGTTTT
168 HIST1 H3G_57040 8355 HIST1H3G CGATTTTTCGTATTAGGCGTTGGAAAGGTAATTTGCGGATTAGTAGTTTA
GTCGATTTTTGATAGCGGCGAATTTCGCGTAGAGTTACGGTGTCGGGAC
GGTAGCGATGAGGTTTT
169 HIST1 H3G_57042 8355 HIST1H3G CGGGTGAAAGTAGGCGGTTTTTAAAAGAGTTTTTTTAAGTTGGATAGAAT
TATTGTTCGGAAATTTTTACGTTTTTTTTTTACGAATGCGGCGAGCGAGTT
GAATGTTT
170 HMG20B_57022 10362 HMG20B TTTTTATGAGCGTTTAGGCGTACGTTGTCGTTTTAGAGGTTGATGTGGAG
TAGGAGGTAGAGGGCGTAGTGAGGGAAATTTGGGTAGGGGTACGTTTC
GGGTAAAAGTTCGGAGGTA
171 HMG20B_57023 10362 HMG20B GAGTCGGTTGAGTAGCGTTGATTTAGGGTGTAGGGCGTGGTTTTTGGAG
GTTTGGGAGGTTGATTTTGTTTTTTTTTTTTTTCGTTAGAAGATTCGAGTTT
172 HMG20B_57033 10362 HMG20B TTATCGCGGGGTATTTTAGTTCGCGGTTTGTTATCGGGTAGTTCGGGCGT
TATTTATTTTCGTTCGCGTTCGTGTTTTAGGTTCGGTTCGGAGCGGTTAT
GTTTTACGGTTT
173 HMG20B_57034 10362 HMG20B GGTGAAAATAGTCGCGGAATTTCGGGTTTTGAGAGGGGGCGGGGGTGT
AGGGGTTTTAGGGTCGGGTCGGGGTTTTTTTTGGTTCGGGGGAAATCGG
TTTAGGTTTTTCGGG
174 HORMAD1_57018 84072 HORMAD1 GGTTAAAAAGTTATTGTCGCGTTTTAGCGGGTGATCGTTGAAGGAAAGC
GTATGCGCGTCGGGTATAGCGCGTGTAGTTTCGTGTA
175 HORMAD1_57019 84072 HORMAD1 TATTGTTTGAGACGTGGCGTTGAGGTTTTTCGTGTTTGAGGTTTTTAGTG CGTTTTGCGTTGCGTTTGGCGGGAAAGGTGATTTCGAT
176 HOXA10_57000 3206 HOXA10 GTTGTTTTATTGCGTTTGTCGTTTAGCGTGGGGAAGAGTTCGTAGTTTTG TAGTTCGTAGGGTAGGTCGGCGGCGGGCGGTAGGTAGATTTCG
177 HOXA10_57002 3206 HOXA10 TTTATTCGGTAAGATCGGGGCGCGTTTAGTTATAGGTTTATGGGCGAGG GTTCGTAGTCGTGCGTTTCGGGATTTAGATT
178 HOXA4_56994 3201 HOXA4 GGACGTGGTTCGTATGTAGGTCGTGCGTTGGGTTTTTGGTTTGCGTCGG
GGGTTGTTCGGGTTGGGGCGGTCGTTCGGGGTTGGCGTCGTCGCGGTA
GTTATAGGG
179 HOXA5_1 3202 HOXA5 GTAGTTCGGGTTATTTGGAT AGCGATCGTAAAATGAGTTTATAAAAT AAG
AGTTT ATTTGTTTTTTGAT ATGTGTGTTTGATTTGTGGTTCGCGGTCGTTT
GTGCGTTTATAGTATTTTT
180 HOXA5_2 3202 HOXA5 TTGTCGGAGTGTATGTTCGTCGAGTTTTTGAATTGTTCGTTTACGGAATTA
TGATTTTTATAATTATGTAATTGGTAGTTCGGGTTATTTGGATAGCG
181 HOXA9_1 3205 HOXA9 GGATTCGTTTTTGTTGGGCGTCGACGTCGCGGATGAGTTGAGCGTTGGT
CGTTATGCGTCGGGGATTTTGGGTTAGTTTTTTCGGTAGGCGGCGACGT
TGGTCGAGTATTTCGAT
182 HOXA9_2 3205 HOXA9 GTTGCGGAGAAAGAT ACGAGGTTTTTGAGTAGGGAAAGTCGAGGTTGTT
ATCGTAGGTTTGGTACGATTAGGGTCGTGATGTTTCGTTCG
183 HOXA9_9564 3205 HOXA9 TAGTTTTTAGTTTAAGGCGACGGTGTTTGGCGTTTCGTGGAATTTAGTGT ACGCGGCGGGCGTTAACGTTGTATTCGTTGCGGTGTATTATTATTA
184 HOXD1_13247 3231 HOXD1 AGGTCGCGTAATTTATTTGGTCGTTGAGGAGGAAAGAGTCGTCGTTCGA
GAAGACGGTCGAGGTGGCGTAGTGGACGTGAGATTCGTCGTCGTCGGT
CGTTCGTTTTAGTACGTTCGGGA
185 HOXD1_13254 3231 HOXD1 TAGCGTTAAGTAAATCGAAAGCGTAGAGGATTTAGAAGAGGGGAAGAGG
AATAATTTTATTTGTTTTGGAGGTGGGCGAAGAGTAGTTTTTATCG
186 HOXD1_13257 3231 HOXD1 TTCGAGAAGACGGTCGAGGTGGCGTAGTGGACGTGAGATTCGTCGTCG
TCGGTCGTTCGTTTTAGTACGTTCGGGAAGTCGTACGTCGGTTCGGATTT
TAGGAAGTCGTAGT
187 HOXD1_56977 3231 HOXD1 TATGTAGTAGTAGCGGCGGGGTCGGCGGCGACGTGTTTAGTTTGGTATT
TAAGTTTTGTCGTTTCGACGTTCGGTTCGTGGTTTTGTAG
188 HOXD1(2) 3231 HOXD1 GTCGGTTGACGTTTTGAGATAAGTCGGAAAAGGGTCGGGTTCGTCGAAG GTCGCGTAATTTATTTGGTCGTTGAGGAGGAAAGAGTCGTCGTTCGAGA AGACGGT
189 HUS1B 135458 HUS1B TGGTGTACGATTTGTTCGTGCGGGTGTTTTTTAGGAGAGTGTGGCGGGA
TTGTTTGTCGTTTAGTTTGCGCGTTTTCGACGCGAGTATTCGTTTGTCGC GTTGGA
190 HUS1B_12677 135458 HUS1B GTTGGGCGGTAGGTAGTTTCGTTATATTTTTTTGGGAAGTATTCGTACGG
GTAGATCGTGTATTACGTTGCGAGCGCGGTTTAGGGACGAGATTAGTT
191 HUS1B_12680 135458 HUS1B GTCGTTTAGTTTGCGCGTTTTCGACGCGAGTATTCGTTTGTCGCGTTGGA
GGACGTTGAGGAGTATCGTGGAGAGGATGGCGAACGTGGGTAGTTAC
192 ICAM1 3383 ICAM1 TAAAGACGTTTTCGCGGTTAAGGTCGAAAGGGGAAGCGAGGAGGTCGTC
GGGGTGAGTGTTTTCGGGTGTAGAGAGAGGACGTCGATTTTTTCGGACG TGGT 193 ICAM1 13230 3383 ICAM1 TAAGTTTAGTTTGGTCGGGAAACGGGAGGCGTGGAGGTCGGGAGTAGTT
TTCGGGGTTATCGTTTTGTTATCGTCGTTCGATTGTTTTAG
194 ICAM5 7087 ICAM5 CGTTTCGGTTTCGTGTTTTTTATCGGGTGTAGGGTTTTTTTTGATTTTTGA
TTTAGTTTCGTTTTTTTTTTGTTTTTGTCGTAAACGTATTTTTTTCGTATTTC GGTATTTTGTTAG
195 ID456952 3400 I D4 GCGAGTAGGGTTTAGGCGTTTTTTTAGTAGTTTAGTCGGGAT AAGGGGG
GCGGTGGAGAGTGAATTTCGGTCGTATATTTTCGTAGTTTT
196 ID456956 3400 ID4 GTTATTTAGGTTGTGGTCGTGTTCGGTTAGGTAGCGTAGCGTTAGTTTTT
CGTCGTCGTAGTTCGACGGCGTTTTGCGGTTCGAGGGGCGTATCGGGTT
TATCGT
197 IGSF4_18983 23705 IGSF4 GATATGGCGAGTGTAGTGTTGTCGAGCGGATTTTAGTGTGCGGCGGTAG
CGGCGGCGGCGGCGTTTTTCGGGTTTCGGTTTCGGTTTTTGTT
198 IGSF4_18987 23705 IGSF4 TCGGATTTCGTTTTTAGCGTATGTTATTAGTATTTTATTAGTTGTTCGTTCG GGTTTCGGAGGTAGTTAACGTCGTTAGTTTGAGGTAGGTGTTC
199 IL17RD 54549 54756 IL17RD TAGGCGTTGATCGTAAAGAAGACGGAGTAGAGTTGTAGTTACGGGGTTA
TGGTCGTGCGTTCGTTTAGTTAGGTCGTTTTTTGCGTTTCGGTCGTTCGT
CGTTGGTTAGTTTCG
200 IL17RD_54550 54756 IL17RD TTTAGTTATAGGTGTCGGCGTTTCGCGCGCGGTCGGATTCGTTAGCGGT
TATAGTTAGTTGCGAGTCGTTGAGGTAGGCGTTGATCGTAAAGAAGACG
GAGTAGAGTTG 201 IL17RD_54556 54756 IL17RD TTTTTGTCGTCGTTAATTCGCGGGGGACGTAGCGCGTATAGGTGTTCGC
GGGGAGGCGAGTTCGCGTTAATTTGTTTGTTTTTCGCGGGGTTCGCGGT
CGGTTTGGGTTTTATTT 202 ING1_24659 3621 ING1 GTTTATTTGCGTTGCGTTCGGGGGGGCGCGGGTAGATCGTTGGTTTGGA
GAGGATTGTGGTAGGTGAGAGGATTTGTGCGTCGTTTTTTGTAGATTTGG
TCGTTTCGGGTGTTAGAG 203 ING1_24663 3621 ING1 GACGAAGAAGGGTTAGAGGAACGGCGTTGTTCGGGATCGAGGTATCGG
GTTCGCGGGAATTTCGGTTTATAGTCGTTTTTTTTTTGTAGTTTCGTTTCG
GCGTTTTATAGTTTTAGG 204 ING1_24679 3621 ING1 TAGGTAGTTAAGCGTGGCGAATATAGTTTTCGGGGTTATTTCGGAGGGG
ATTCGCGGCGGTCGTAGCGGGGCGGAGGTCGAAGTCGAGGTCGTTTCG
GAATTAGAGAGT 205 ING1_24685 3621 ING1 TCGAAATAGAATTGGTAATCGTAGTAGTTAATTTATTTGTTAATATTATTTT
GATAGTTTAATTACGATGAAGAGGAGGCG 206 INHBB_56906 3625 INHBB GTATTTCGTGTCGCGGTTCGGCGCGGGGACGTCGTCGTCGTTCGTTCGT
TTTTTTTCGGTTTTGCGGCGGGTGTTAGTACGAGCGCGAGCGAGGGTAT TGTATTCGG 207 INHBB_56909 3625 INHBB CGTTCGTATTTGAGCGTTTCGTTTTTATTGTTTCGCGTTAGGAAGGGTCG
TCGGCGGCGGAGACGGGTAGGTCGTTTTCG 208 IRF7_18344 3665 IRF7 GTTAGGTCGCGGTTATAGGTCGTGTGGTTAGGTGTTATAGGTGTTTATAG GTGTGGATTGAGGGTTTGTAGTTATCGACGTTGTTTCGGTATG 209 IRF7_18346 3665 IRF7 AGTTGAGAATCGGACGGGGTGGGATCGAGGAGGGTGCGAAGCGTTATT GTTTAGGTTTCGTTTTTTCGGGAGTTTGATTCGTT 210 ITIH5_56885 80760 ITIH5 TCGTGATAGTGAGTGAGTTTTTACGAGATTTTATGGTTTAAAAGTTCGGC GTTTTTTAGTTTTGTTTTTCGTTTTTGTCGTTTTGTAGACGTGTTCGTT 211 ITIH5_56887 80760 ITIH5 CGTTGTAAAGCGTGTTTCGTCGGGTTTTCGAGCGTTTCGCGTTTTCGTTT
CGTTATGTTTTTGTTGTTGGGGTTGTGTTTGGGGTTGTTTTTGTGTGTGG
GGTCGTAGGAAGAGGCGTAG
212 KCNG3 56878 170850 KCNG3 GAGGGTTGCGTATATCGAGGTCGCGGTGTTTTTTTTTAAGTCGCGGGGT
CGATTTTTTGAGGGTTGCGGGCGTCGAATGGAGTCGTCGGGGCGGAAT
AGTTTTTCGT
213 KCNG3 56880 170850 KCNG3 CGTTGTTAGTAGCGCGTTTTTCGTTCGGCGGTATTTGCGGGTGGTCGGG
GAGTTTTCGTCGGCGTTAGCGTTGAGTTTTATCGGTTG
214 KIF1AJ 547 KIF1A TTTTTATTTTTCGCGGGGTTCGTCGCGGTGTTAGCGGTGACGTTATAGTT
CGGTTTCGGTTTTTTTCGTTTTTAGTTTTATTTTCGCGGCGGTCGCGTCGT
TCGTTTTTTTTTTCGCGCGATAAATTAGTTGGCGATT
215 KIF1A_2 547 KIF1A GAGGTTTTAGGGGTACGTCGGGGGAGGGGACGTTGCGTTCGAGTCGGG
TGTCGGTTTTTTATTTTTCGCGGGGTTCGTCGCGGTGTTAGCGGTGACGT
TATAGTTCGGTTTCGGTTT
216 KL_56871 9365 KL GAATGAATTTGAGCGTTTACGAAACGTTTTGTACGGTTTTCGGGAGTTGG
GAGAAATAGGTGTTTTTTTTCGACGTTCGCGGGCGACGTTTGTCGTATTT
217 KL 56874 9365 KL AGATGGACGTATTTTTGTCGTGTTGTTGTTAGTCGTTTTCGGTTTGGTAG GCGGCGTTGTTTACGGTTTAGAGGAAGTCGTC
218 KLF42 9314 KLF4 ATTAGTAAGGCGAGTAAGTAGGTTCGGTGGTCGGGTTGCGTTTTTTTTTA
TTTAGTAGCGTTTTTTATTATTGTCGCGGTCGTTTCGAGTGTTGTCGTGG G
219 KLF4 24636 9314 KLF4 TTTGTTTAATTGCGTGTGAGCGAGCGTCGCGGTTGGTTTTTTTTTTTTTAG
GTTTCGTGGACGTTTTCGGAATTGGTATAT
220 KLF4 24642 9314 KLF4 TTTTCGGGATAGTTTTCGTGTTTTTGGTTTAGTTGTGTATGTTCGTGGTGC
GAGTATTGCGGAGTCGTTTGGGGCGTAAGTCGGGTTTTTATTA 221 KRT14_56851 3861 KRT14 TTGTTGAAGTTATCGTTATAGTCGTTTTTTAGTTCGTAGGTTTTTTTAGAG GAGAAGCGGGAGGATGAGATAGATAGGTC
222 KRT14_56852 3861 KRT14 GTTAAGTTTAGTGCGCGGGTTTTTGAGGGTTGGGATTTTTAGGGTTCGAT
GGGAAAGTGTAGTTTGTAGGTTTATATTTTTTTTTGTGAATTACGTTTGGC
GGGATAAGAAA
223 KRTCAP3_56842 200634 KRTCAP3 GTTGATGCGTGTGGGTTTCGCGTTGATTTTGGTGGGTTACGTGAATTTGT
TGTTGGGGGTCGTGTTGTATGGTATCGTTTTGCGGTACGTGGTTAATTTT
224 KRTCAP3_56843 200634 KRTCAP3 TACGGGTATTAGGGGAGTCGAATTTTATAATTTCGTTGGGGCGGTCGGG
GCGGGAGAGAAAGGTGGTGTTGTAGTGGTGGTTTTGGGGGGTTATTCGA
TTCGTTTTAGTTGTTGTT
225 LEPR_56815 3953 LEPR TATATTTTAAGGGACGAAGGGAGCGGTCGATCGTTTTTCGAATCGGAGA
GGCGGGAAGGGTGGTGAGGTCGTTTTTTAACGGCGCGCGGAAGGCGTA
GTTTTAG
226 LEPR_56822 3953 LEPR TTTGGAGTCGTAGGCGTTGTTTTCGCGAGGTAGGGGAGGAGTTTTGTAT
TGTTAGGGGCGGGGTTTTGAGTAGCGAGGTCGGGTTTGAAT
227 LEPR 56824 3953 LEPR TTAAGTTAGATCGGGAGTCGTTTCGCGGATTTCGAGTTCGTTTCGGTTAC GTAGAGTTTCGTTTTCGTCGGTGTAGAATTTCGATTTTAGT
228 LGR6 59352 LGR6 GTAGGGGTACGGTTAGGCGGGTTGGGGTGGGTAGGGGCGCGCGCGCG AGGTTGGTTTTCGTTTTCGGGGTCGTCGTTTAGTTTTCGGGTACGTGC 229 LGR6_56719 59352 LGR6 TAGAT ATTGCGTAGTAGGCGGGGTATAGAGGAGGGAACGTTTGTGTTTA ATTGATGTTTTTAATGCGGAAGTTTTTGAAAGGCG 230 LGR6_56730 59352 LG R6 TTTTGATTGTATCGTTTCGGCGTTTTTATCGTCGTCGTCGTCGTTTAATAG AGTTTTTGGGGCGGTTTTTATCGACGGTGTAGTTCGTCGGGAT 231 LGR6_56731 59352 LGR6 GTTCGTAGGATTAGGCGGGGATAGGCGGGTAGTATTTATAGGTAAGTCG TTAGGGGGTTTAGGTTTTTCGGAACGGCGGATAGTTCGAG 232 LHX6_23427 26468 LHX6 CGTTCGTTATGGTGAGTTCGTTTGGTTTTGTTTGCGGTGTTTAGTTTTTTA
GCGGTTCGGTTTTGGGGATTTGGTTAGGTTTTTCGGTTACGAATTGTTGT
TTACG 233 LHX6_23428 26468 LHX6 GTTTTGTCGTTGGAGTTTCGTTCGGTTTAAGATGAGTTATGCGTGATTAAT
TTTTTTTTCGTTAAGGGAGAGTAAGAT ATATACGACGAGGTAGAGATTTA
GAGAGA 234 LHX6_23430 26468 LHX6 TTGGGTTTCGGATTTTGTTCGATTTCGGTTCGGGTCGTTTATTTAGTCGTT
CGTCGGTTTATTTGGTCGGTGGCGGGGTCGTTTTCGGTCGGTAGTCGGT
AGTT 235 LIPG_56689 LIPG CGTTGTATATTTATCGCGTGGACGTCGTACGTTCGGTTTCGTTTTTTTTCG
GGTTCGTTTCGTAGGTAGTTGCGATTTTAG 236 LIPG_56693 9388 LIPG ATTTGGCGGTACGGAGTTCGGGCGGTGGTAGGGAGCGAGTGTTAACGG TTTGTTTTAGAACGAGATTTGTTTTCGTTTTGAG 237 LOX_18977 4015 LOX GGTAGAGGCGAGGAGTTGTTCGTTTTGTACGTTTTTAATCGTATTACGTG AAT AAATAGTTGAGGGGCGGTCGGGTTAGAACGGTTTGTGTA 238 LOX_23395 4015 LOX GTTAGATTGATTTCGTTCGAGGAGGACGTGGTTTATAGAAAATAAAAACG
GGGTTTAAATTACGTGAGGGAAGGAGAAATTTTTAATTAAGGAGGCGAG
CGGAGTACGGGTATTTTAGTT
239 LSMD1_56678 84316 LSMD1 GTTATATTTCGCGTAGGATTCGGACGGAGAGCGCGAGGATTCGGCGGTT
GAGCGCGTTCGATAGTAGTTAGAGGCGTTGT
240 LSMD1_56679 84316 LSMD1 GAGGTTTTCGGGTAAATGGTCGGAGTTGGATCGATTATGTTGTTACGAGA AGAGAATGGTTGTTGTAGTCGGCGTTAGAGTAGTTTTAGTG 241 LSMD1_56683 84316 LSMD1 ATTTTTATATGTTTTACGCGGGATTTATATTACGTTTTTCGTGAATACGTGT AGTTT AAATTTCGTTTTTGAT ATTTATTTTAGTGGACGGTGGTCGGAAAAG GATA
242 LTB4R_31247 1241 LTB4R GTAGTTCGGTTTTGGTTCGTCGTTTGTTGTTGGCGGTTTGGTTGGTCGTT TTGTTGTTCGTCGTTTCGGTCGTCGTTTATCGTTATTTGTGGAGGGATC 243 LTB4R_31250 1241 LTB4R TAGTAGATTTTTAGCGGTGAAGACGTAGAGTATCGGGTTGACGTTAGAAT
TGAAGAAGGTTAAGGTCGTAGTTTTCGTTCGCGTCGTTTGGTCGGTTTCG
TTTAGTTTCGTTAAGGTTTT 244 LTB4R_31257 1241 LTB4R TTTAGTTCGGTTGAGTTAGAGGCGTAGTTTTCGGGTCGGGGATGAGCGC
GAAGTTTGGAGATTTT AAGTTTTAAGTGGGACGAGCGTGCGTCGTTTATT
TGTTATTTG 245 LTB4R_31261 1241 LTB4R TTTTAATGTTGATAGCGTTCGGCGGAAATATTTTAGAGTTTTCGGGTTTAG
GTGCGGGTGCGAATTTGGAGTCGGTTTCGAGCGTTTATTCGGAGGCGGT
TAG 246 LY6K 54742 LY6K GCGGGGTTTTTTTTATCGGTTAGATTCGGGGAGAGGCGCGCGGAGGTTG
CGAAGGTTTTAGAAGGGCGGGGAGGGGGCGTCGCGCGTTGATTTTTTTT
GGGTATCGTTG 247 LY6K_12884 54742 LY6K TTTATCGGTTAGATTCGGGGAGAGGCGCGCGGAGGTTGCGAAGGTTTTA
GAAGGGCGGGGAGGGGGCGTCGCGCGTTGATTTTTTTTGGGTATCGTT
GGGGACGATGGCGTTGTTCGTT 248 LY6K 12885 54742 LY6K GCGTATTAGTGTGTGAGGTTACGGTGTTTGCGTGTTTTGAGGATTTTTTA
AAACGTGTTTATTAGTAAGGTTTTTGTTTGGGTCGTTCGTGAGTAATTTTG
TA 249 LZTFL1_56661 54585 LZTFL1 GTCGGTTATTTATTATGGCGGTAGGTAGCGGCGGTAGTTTAAAGGAACG GGAGAGGTTAGGCGGTGTTTCGTTAAGTTTGGGATCGT
250 LZTFL1_56669 54585 LZTFL1 ATGCGTATGTGGTAGTTAATCGGTTACGTAATATTTTTTGTCGCGTGATTT TGAGGATTAATGAAAATGTATAATTGTAGTTTACGTGTTTCGGTTTTCG
251 MAGEH1_57803 28986 MAGEH1 CGAGAGTCGTAGAAGAGAATCGTAATAATCGTAAAATTTAGGTTTTAGAG GTTTTCGAGATTTTTATGGTCGTTTTTGTGGTAGCG
252 MAGEH1_57810 28986 MAGEH1 GTCGTTTTCGGGGGTGTTCGTTATTATAGAGGCGGTTATAGGGGTTTCG GAGGTTTTTGAGGTTTGGATTTTGCGATTGTTGCGGTTTT
253 MAL_1 4118 MAL TTCGGGTTTTTTTGTTTTTAATTCGCGCGCGGGGGCGTTTAGGTTATTGG
GTTTCGCGGAGTTAGCGAGAGGTTTGCGCGGAGTTTGAGCGGCGTTCG
TTTCGTTTTAAGGTCGACGTTAGTACGTCGTTATGGTTTTC
254 MAL_56656 4118 MAL GTGGCGGTGGTTTAGTTTCGTTAGGAAATCGTCGTTTGGAGTTGTGGGT
CGCGTATATTAACGTATTTAGCGGAAAAATGAA
255 MAL_56660 4118 MAL TTTTGGTTTAGTTTTCGTTTTGCGTTTGTTGTTTCGATGTTTTTAGAATTTG
CGTAGTTAGCGGATTTCGTTCGATAGTTGTGTTGGGCGTTCGAGAGAGC
GTAC
256 MET_54299 4233 MET GTTAGTTTTTATTTTAGCGGTTTTCGGAATTCGCGGATTAGGGGACGGAT
AGTACGCGAGGTAGATAGATACGTGTTGGG
257 MET 54300 4233 MET TAGCGGCGTAAGGATTATACGCGCGTTTCGCGTTTTTTCGTTTTTTTTTAG TAAGTTAGTTGTCGTTTCGTATTTGGTTCG
258 MGC33846_56643 220382 MGC33846 GTTTATGAGGTCGTTGTAGCGTTTGATTTGTTTTTGTAGGTATTTGCGGT GGTTTATTTTTCGTTTCGATTTGTTCGGTTTGTT 259 MGC33846_56649 220382 MGC33846 GGTAGCGTTGCGGATAGTTCGGGAGTCGCGGCGATGGCGGTGTAGGCG GCGTTTTTTAGTACGTATTTTTTCGTGTTTTTCGGTTTCGG 260 MMP2 4313 MMP2 TATCGAGTTAGCGATTTTCGGGCGACGCGCGGGGTTAGGGAGCGTTAC GATGGAGGCGTTAATGGTTCGGGGCGCGTTTACGGGTTTTTTGAGGGCG 261 MMP2_52612 4313 MMP2 GGGTCGGATTATGAGTCGTTGAGTCGGGTAAATTTTAGGTTATCGAGTTA GCGGATTTTCGGAGCGTAGTTTTGCGTCGCGGAGTAGGTTTTAATT 262 MYCL1_54211 4610 MYCL1 GCGTTCGTGTTTTGGTTATTCGTAGTTTTATTTCGTTTTAGTCGTTCGTTA
TTTGGAGCGGATCGGTTTTTCGTCGGTTCGGGGTAGTTCGGTAGTTAGT
AT 263 MYCL1_54212 4610 MYCL1 GATTCGATTGTGGGTAGCGAGTTTAAAGTAAATTTTGTTAGCGTCGTTCG
GAGCGTAGTTTTTAGGGTTCGGCGGGGTCGGGCGGGGGCGCGTCGTGT
TTAGAAGGTAGTTT 264 MYCL1_54213 4610 MYCL1 GTAGTTTGCGTTTAGTTTTCGCGTTTCGGGAAGTCGGGTTTCGGGTTAGA
GTGGTAGGGGAAGTTAATCGTAGCGCGCGGATTCGATTGTGGGTAGCG
AGTTTA 265 NAALAD2_56618 10003 NAALAD2 TTTGCGAAGGTTAGCGGAGGTTATTTAGAGTTTATAGTTTTTTGTTAGCG
CGTTTTTTGTTTTTTTGTAGTTTCGAAGTTCGCGAATGTAGTAGGCGTTTT
AAGTTCGGTTTTTAAG 266 NAALAD2_56623 10003 NAALAD2 AAGGTATAGACGGTTTTTGGATTTCGTTATGGTTTTTTGAGGATCGAGTTT
GGGGCGTTTGTTATATTCGCGAGTTTCGGGGTTGTAGAGAAATAGAGAG
C
267 NAGS_57828 162417 NAGS GGGTGTCGAGTTTTAGGTTTTCGGTGTTTTACGAGTTTTTAGAGTTTTTTT
CGGGTCGTTCGTTGGTGTAGCGGGATATTTA 268 NAGS_57831 162417 NAGS GGATTCGGTATTTACGACGGTTTTTCGGCGACGGGCGATTGGGAGACGT
CGTTCGCGTTCGCGTATTTTTCGGGCGGGGGTTGGGGTTGCGATTAGGC
GGTGTTGAGT 269 NDP 1 4693 NDP TTTCGTGGAAGGGAGTCGAGCGGTGGGTAGAGGTTGAGTTTTCGATAAC
GAGCGTTTTATATTTTCGTGGTATTTTTATTTGTTAGTGCG
270 NDP_8597 4693 NDP GTTACGGAAATGTGAGGCGTTCGTTATCGGGGATTTAGTTTTTGTTTATC GTTCGGTTTTTTTTTACGGAGTATTTTACGATGGGAATAG
271 NDRG2_56603 57447 NDRG2 AGATTTTGTGGTTTCGTCGTTAATTTTTTTTAGTTCGGTTTAGAATAGGAG ATTAGTTTAGGTTCGTTGAATCGTAATGTTCGGGGGAT
272 NDRG2_56609 57447 NDRG2 TATCGAGAGGGACGCGGTAGATTTTGAGATTATTTTTCGGGTATTGCGAT
TTAGCGGGTTTGAGTTGGTTTTTTATTTTGGGTCGGATTGGGAGGGGTTA
GCGGCGAAGTTATAGGGT
273 NEDD4L_56593 23327 NEDD4L AGAGAGGTAGGTATTCGCGGTAGTAGTTTTTAATTTAGATAGGTTTAAGT
CGCGCGAGGGTGTTCGGGTGGGTGGTTGCGTAGGGTTTTATTTGGGC
274 NEDD4L_56595 23327 NEDD4L ATAATAGTAACGGCGGCGAGGAGAAATTTTGGTTTTCGTTAGTTTTCGGT TAGGTAATTAATAGTTTGGAGTGGGAGGTGTCGTGTTGGGGAGGTATC
275 NEDD4L_56599 23327 NEDD4L CGTGGGGTTGAATTTCGTTATTTTCGTAGTTCGCGGTTCGTTCGTTAGTT
CGGTTTGTGATTGGTTTGGGGGGCGCGTGTGTTCGTTTCGGCGAACGG
GGAGGGTATTTTC
276 NEDD4L_56600 23327 NEDD4L TTGTAGACGTTGGGTACGAGTATTTCGGGGACGGTTTTTTTTTTTTTTTTT
TGTCGGCGCGGGATAGGAATTTTTCGGGGAGTTTCGAGTAG
277 NEF3 13215 4741 NEF3 TGAGTTATACGTTGGATTCGTTGGGTAATTCGTTCGTTTATCGGCGGGTA ATCGAGATTCGTTCGAGTTTTAGTCGCGTTAGCGGTTTTT 278 NEF3_13218 NEF3 ATTATTGGGTCGGGGAAGCGTTGTTTGAACGGCGGGATAGTGATAAAGA AAGGGCGTTGGCGATATTCGTATTAAGGGTGCGAAACGTAATCGGGA
279 NEF3_13224 4741 NEF3 TATTATTTTTAGGGTGGCGCGTAGTTCGCGGATTTTTTGGTCGTACGCGT CGTTTAGTTGGGCGTGCGAGGTTTGTTTTTGTCGTAGCGTTTGGATTTT
280 NEFH_18450 4744 NEFH TATGTCGAGGTAGAGAAATCGGTTTTGTAGTTTT AGGGTTTCGGTTTTTTT TTTTGGAGAGTGTTCGTTTGTGATAGGGTTTCG
281 NEFH_18452 NEFH GTCGGATGAAGTATTCGGGCGTTTTTATTGCGGAAGGGCGGGGATGGTT GTGACGTAGGCGTGTTCGTCGTCGTTTGTAGGG
282 NEURL 9148 NEURL GAGCGTTTAGAACGTTTCGCGTTTCGTCGAGTTTCGTTTTACGTAGATTC
GCGGGCGGGAGGGAGTTACGTATATCGTCGTCGCGGTCGTTTTCGCGG
GGCGGTAATCGAGTTTGTTTCGGAGTCGTCGAACGTTTACGTTAGCGATT
TT
283 NEURL_13058 9148 NEURL GTTGTTATGGTGATAGTTGCGTCGGTGAATGAGCGTTTTAGTGATACGCG
CGCGTTCGTTAGGTTAGATGTGGTTTCGGGGCGCGTCGCGTTTTCGTAG
TCGTAAGTTTTCGGC
284 NEURL_56560 9148 NEURL CGGTTTTGTTATGTCGGTTTTCGTTCGCGGGGGAGGAGACGGGGGAGG
AGGAGGGGAGTAGTCGGTCGGGGCGATTCGGTGATTAGT
285 NEURL_56564 9148 NEURL AGGTATTCGCGGGGTTTTATCGTGGAGAGTGTTTCGGGTGATTTGTTTTT TTATTTTCGGCGACGGGTAGTGTAGTTATT
286 NPPB_13212 4879 NPPB GGAGTACGGGGTGATTTATAGCGAAAATAGTTTCGGTTAAGATTTCGTTA
TTCGCGGCGTTTTTTTTTTTTTCGCGCGAGTTTTTTGGTTCGTACGTAGC
GTAGTATTTGTTCGTCG
287 NPPB_13214 4879 NPPB TAGGGTGTATAGCGGCGAGTAGGTGTTGCGTTACGTGCGGGTTAGGGA
ATTCGCGCGGGGAGGGGAGAGGCGTCGCGGGTGGCGGGGTTTTGGTC
GGGGTTGTTTTCGTTGT
288 NPPB(2) 4879 NPPB GGGGATTTGTTTGTGTTTTTAGCGCGTTTTTGTTTTTCGGTTCGATTCGGT
TTATTTTTATATAAGGTCGGTTTTGTTCGGTTTTTATTTTTTACGTC
289 NPTX2_57773 4885 NPTX2 TTTTAGTTTGTGACGTTCGCGTTTTCGGGTGGGTTCGAGGGGCGTTTGG GTACGGTTAGTCGAGGTTTTCGAGAGTTTTA
290 NPTX2_57779 4885 NPTX2 GCGTCGTTTTGTATGGGTATCGCGGGTAGCGGGTAGTCGGCGTGTATCG TTTTTGGGGGTAGTGTCGTGTATACGAAGCGGTTATCGGG
291 OGDHL 55753 OGDHL TCGTTAGTATCGTGGATAGCGTTAGGAGCGGGGCGGGGTATTTATGGAG
ACGTTACGACGCGCGGAGTTTTAGGGGCGCGCGTTCGTGGGCGGTTGC
GTTACGTGATTTAGGTAATTGGTACGGCGCGCGCGTATTTTTTTCGCGTA
GTTTTTTGATTTGTA
292 OGDHL 19631 55753 OGDHL GTTTTAGTTTCGTTTTGCGGTTTTAGAGAGGAGTTTTTTTTTCGGTCGTGG
GCGATTTCGGGTTTGGGTCGTTAGTATCGTGGATAGCGTTAGGAGC
293 P14 1029 CDKN2A GTGTTAAAGGGCGGCGTAGCGGTTGTCGAGTTCGGTTTTGGAGGCGGC
GAGAATATGGTGCGTAGGTTTTTGGTGATTTTTCGGATTCGGCGCGCGT
GCGGTTCGTCGCGAGTGAGGGTTTT
294 P16 1029 CDKN2A TTATTAGAGGGTGGGGCGGATCGCGTGCGTTCGGCGGTTGCGGAGAGG
GGGAGAGTAGGTAGCGGGCGGCGGGGAGTAGTATGGAGTCGGCGGCG
GGGAGTAGTATGGAGTTTTCGGTTGATTGGTTGGTTACGGTCGCGGTTC
GGGGTC
295 PAX3_18479 5077 PAX3 TTTGGGTATAGCGTCGGTTAGCGTGGTTATTTTGGGGGTAGTTTCGTTCG
GAAATTATATTTAGGTGAAGGCGAAACGGAAAGGCGAGTGCGGCGCGG
ATGATTTTCGGGAAT
296 PAX3_18482 5077 PAX3 TTTGGGTAAGGGGCGTAGCGCGGGTTTTTTTCGGGGTTAGTAGAGGTTT
CGGTATTATTAGAGATGGGAAGAGAAAGTGGTCGTTGTTGTTTAATTAGC
GCGTGTTTTCGTTATTCGGG
297 PCSK6_56480 5046 PCSK6 TCGGGTCGTTTAGTATTTGTATCGTTTAGTGGTTGGTGTAGACGGGGCG
CGGCGGGGGCGGAGTAGGCGGTAGGTAGCGTTAG
298 PCSK6_56483 5046 PCSK6 ATAGGATTGCGGAGGCGTTTTTTTCGCGCGCGTCGGAGGTCGTCGGGA AAGTTTTGGGCGCGCGGGGTCGGTCGTATTTATTTGGT
299 PDLI M3_18531 27295 PDLIM3 GGAGGTATTCGAGTTTGGTAGTTTCGTTTTTTTTGAGTTTAGTGGTTTTTT TCGGAAAGTCGTTGCGGATTTAGAGGATG
300 PDLI M3_18532 27295 PDLIM3 AGGTGTGATTTTAAGAGAGCGACGGACGTTGTAATTCGAAGTTTTCGCGT TCGTCGCGGTTCGGGAGGTTGTTTTAGTCGGTCGGGTTGCGTATTTTC
301 PIK3CA_66163 5290 PIK3CA AAAAATAGGGGCGACGGAGAAAGGAGTCGGGGGCGGGGGCGTGTGGC
GGGGGTTAGCGAGGAGAGGGAGCGAGAAGTAGAAAGCGGTAGTTTCGG
TGTC
302 PIK3CA_66165 5290 PIK3CA ATAGAGTTATCGCGGTCGGGGCGGCGGTTTTGGGCGTATTTTTTTAATTT
TTAGTCGTTTCGTTTTT AAAATTGGGAGTAGAAGTAAAGGAAGAGCGAGG
CGGTAGTTAGAG
303 PLAGL1_52944 5325 PLAGL 1 CGTTCGTTCGTTTAGTATTCGTTCGGAGAGTGAGGTTAGAGTACGTTTTA
GTCGTGTTTAAATTAAGGTTCGGGGCGGTATCGACGGGTTGAATGATAA
AT
304 PLAGL1 52949 5325 PLAGL1 CGTTGTAGATTTTAGGTCGGTTCGGGTTTATTTGCGTTAGCGTTGTATTT
GGGCGATTTTGGTTTTGTTTTTATCGGTGATTCGGTTCGTAGGACGTGTG
GGTGTCGTTTAG 305 PLAlM 8620 5328 PLAU GTAGTATAGTGCGGAGATCGTAGTTTCGGAGTTCGGGTTAGGGTTTATTT
GTTTTCGTAGCGTCGGTTCGCGTTTTTTTGTCGTAGTTATCGGTGAGTGT
CGCGGTTTTGAGATTT
306 PLAlM 8623 5328 PLAU TAGGGTAAGAGGCGGAGGAACGGGAAGGTAGGTTAGGCGGGCGATTGT
AGCGTAGGGGAGATGTTCGCGGTGATTAGGTTTT
307 PLXNA4B_57788 91584 PLXNA4B CGTTCGTTCGGTTTTATTTTTCGGCGGGTCGGTTTTTTATTTGGACGCGT CGCGTTTTTTTTTTTTAGCGGGAGCGTCGTATTGATATTGTAGATG
308 PLXNA4B_57794 91584 PLXNA4B TTGGTATTAAGGATATTTAGTTCGCGGGGGTGGGGACGAAGGGGGGTA GTTCGGCGTCGTTATCGTAGTTTTCGAGAGTC
309 PNMA3_56433 29944 PNMA3 TTTTTCGTTTCGTTGGTATTTTCGTGGTACGTGCGGGAGGGGTAGGAGT
CGGGATTAGGAATCGTTGTGGTGAGAGTTAAAGGTACGGGGATTGGTCG
TTATAG
310 PNMA3_56439 29944 PNMA3 TTTGTGAATGCGAGTAGGGCGAGAGCGAGGTTAATGTTTATTTTTTCGTA
CGTTTATATTTACGTTTATTTTTATTTTTATTTTTAGTCGTTTTTTTTTTCGT
GCGGCGTTTTTGTAGT
311 PON2_56425 5445 PON2 TAGGTTTAGGGCGGAGTTTTCGCGGTTTCGACGTAGGGATTCGGTTTAG
GCGGAGGACGGGGCGGAGCGCGGTCGGTATTATCGAGTCGGGAAGAT
GGTATCGT
312 POU2AF1_23443 5450 POU2AF1 TTGTTATTTTATTGTGCGCGTTTTAGCGGTTTCGGTTTAGAGCGTTTTATA
TTCGGAGTTAGTTAGTTGGGATAGAGTAATAGCGTAGCGCGGGTAGTTA
TAGTTTG
313 POU2AF1_23446 5450 POU2AF1 TTTTGGGAATCGTATTTTTCGTTGAGTAGGCGTTTATGGCGGGTTGAGTT
TTGTTTATGTTTTGGTGGTTTGGAAGTTTGTATGGGCGTCGTGTAAAGAT
TAAC
314 PPP1 R13B_23470 23368 PPP1 R13B GTTCGGTTTCGTATCGGTTCGCGGGGGTTGGGAGTCGCGGGAGGAAGG
AGGTTTGGTTAGCGGTTCGCGGAGGAAGTCGTCGTTATT
315 PPP1 R13B_56416 23368 PPP1 R13B GTTCGTCGTAGTTGTCGTTTCGTCGCGGTCGGGTCGGAGAGTACGGCG GCGGGAGCGCGGTTTTAGGAGGCGGTCGGAGCGGTGGGTATAGTTCG
316 PPP1 R14A_8724 94274 PPP1 R14A CGTTATCGTTAGAGGGCGTAGATAGGTCGGGTTTCGAGTGTTTGTTTTAG GTTTTCGTTTTATAGGATTTCGGTTCGGGAGTTTTCGATGTAG
317 PPP1 R14A_8725 94274 PPP1 R14A CGTTATTGTATTTTAGTTTGGGCGATAAAGAGAGATATCGATTTAAAAAAA
AAAAAATTCGTTGAACGGAATGAGTGAATAAATAAATGGATGGATGAGTG
AATGAATGAACGAAGGAATAAGTGATCGT
318 PROX1_56401 5629 PROX1 TTTTTCGGTAGAGAGAGGAAAATACGCGAGTGTATATACGGTGATATTTT
GCGGGTGATGAATATGATAATTGGATAAGGGACGGTGAGTTTGTGCGAT
A
319 PTEN_10192 5728 PTEN GAAGAGGAGGCGAGAAACGTCGTCGTTGTCGTCGTCGTAGGTCGGTCG
GTTTTTCGAGGGCGTTGTTTTCGCGGTTGTTTATAGGCGTTGAG
320 PTEN_9406 5728 PTEN TAAACGAGTCGAGTTATCGGGGAAGCGAGAGGTGGGGCGTTGTAAGGG AGTCGGATGAGGTGAT AT ACGTTGGCGAT AT AAT AGTAGGTTG
321 PTPRD_56390 5789 PTPRD TTTTTAGTATTCGTGCGGAGGAAGAAAGTGAGGAGTAGTAGTAGTAGTTT
GGTTACGTGTATTATTTTGTAGTTTGGTAGTAGCGTGCGCGAGTAGTTTG
GAATTATTGTTTTC
322 PYCARD_52546 29108 PYCARD GAATAGAAAGCGGAAGAGTTCGCGGGGTAAGCGTTGGTGTGGGTGGAG
GGGAAAGACGGGGTGGAGGGGAACGGGGGCGGTTTATTTTGGTGCGTG
GTCGTTTGTAGTTGTTCGGTTATT
323 PYCARD 56380 29108 PYCARD AGTCGTCGATTAGGAGGAAGTCGGTTTCGGGGCGGAATTTGGATTTTTC
GTTTTTTTTTTATTTTGGTTTTTCGATTTTTCGTTTCGGTTCGTTGT
324 RAR2β 5915 RARB GGGATTAGAATTTTTTTATGCGAGTTGTTTGAGGATTGGGATGTCGAGAA CGCGAGCGATTCGAGTAGGGTTTGTTTGGGTATCGTCGGGGTA
325 RARB_23618 5915 RARB TTTTAGTAGGATTTATTTGTCGGCGACGGGGTGTTGTATTTATTCGGAGG
CGGATGTTTATGAAGGTTATGGAGGGGAGGTAGGGAAAGTTTTTCGATG
ATAGGTGGAAAGAGTAAC
326 RARRES1_57577 5918 RARRES1 GTCGTTTGTTGTAGGAGTTTGCGCGGGATTTTAGTATTTTGAGGTTGTTT
AGGGTCGTCGGGGTTTTCGGATTTCGCGGGCGTCGTTATCGGGGCGAG
TAATAGTAGTAG
327 RARRES1_57581 5918 RARRES1 GCGAAATCGTAGGGGAAACGTTAAGTTTTTGAGTTGGGTTAGGGACGCG
GTACGAGGTAGTACGGGCGGGTCGCGATTTTTCGGGGAGGCGTTTTTAT
328 RARRES2_57694 5919 RARRES2 TTGGGATCGGAGAGCGATAGATGTGGAAATCGAGGTTTTTTAGTGAAGA
GTTGTTAGGGTGGTCGTTTTAGAGTAAAGGCGTTTTTTATTTCGTTTGCG
TAGTTGTTTTT
329 RARRES2_57700 5919 RARRES2 AGTAGAGTAGCGGAGAAGAGGTTCGGGTCGGTCGAGGTTGCGGAATGT
TTAGGTTCGAGTTAGAGGGAGGTGGGATCGAGGGTTGCGCGTTTGGGC
GAGTCGTTTGTAGTC
330 RASSF1A 1 1186 RASSF1 GCGTTGAAGTCGGGGTTCGTTTTGTGGTTTCGTTCGGTTCGCGTTTGTTA
GCGTTTAAAGTTAGCGAAGTACGGG
331 RASSF1_8476 1 1186 RASSF1 TCGTTTTTAGGAATGATTTTATCGTTTCGGAGTTTTATTTAT AGATTTTATT
TATTATAGGGAACGGGGGCGGGTGTTAGCGTTCGGGTAAGCGTATAAGA
GTG
332 RASSF1 8480 11 186 RASSF1 GTTTGCGAGTTAGCGAGGTTCGCGCGGTGAAGTATTGTTCGAGTTTCGA
GTTCGAGTTTTTTTGGTTGTAGTAGTTATTGTTCGTCGTGTTGTTTTAGGT
TA 333 REC8L1 13013 9985 REC8L1 GCGTTTTTGTTGTTAGGTTTGCGGCGGGGGAGCGGCGGGGGAGCGACG
GGGATGCGTTTATTGGTTAAGGAAGGGGCGTTTGTTATTAGAGGCGAGA ATCGGAGTTTATT
334 REC8L1 (3) 9985 REC8L1 CGATTTAGTTTTGTAGTAGGCGGTTCGGGGTTATATCGCGGTCGTTTAAG
TTAGTGTAAGGTTTAGGGGTTTGATATCGTTTTTAGCGTTCGAGGATC
335 RP11-450P7 3_56363 257240 RP11 -450P7 3 CGTGTGAGTTAGGTTAAAGCGCGTTGTTACGTTGGCGGCGAAGTAGTAG TTTTTTGGAGTTAGTTGGCGTTTTAAGTAGCGTTTATA
336 RP11-450P7 3_56369 257240 RP11 -450P7 3 TTATCGTCGTTTTTGGCGTGTAGGTGTGGGTTTTTTATATTGGGTCGGGG
AGGGGATTTCGGGTAACGCGTTGGAAGTTTCGGTAGTCGTGGAAATTAA
GT
337 RP11-450P7 3_56371 257240 RP11 -450P7 3 TTTTTAGTGAATTTCGGTTTTCGTAATTTTAATCGGGTTTTTGGGATTTTTT
GGTGATATTGGTGGTTAGTAGGGTTATTGGAGTTAAGTTTTTGGTTCGGG
TCGTTTTATTCG
338 RPL10_56355 6134 RPL10 GTCGTTCGGAAATAAGATAGAGAACGTATATAAAGGTTTTAAGAGCGTAT
TTAGCGTTCGAAAATGCGGTGAGTTTGTAG
339 RPL10_56356 6134 RPL10 TTTTTCGGGTTGATAAAGGAACGCGGGTTTAGATTTTCGTATGGCGTTTT
ATCGTCGGGGTTTTTAGTTTAGAAGGAGGTACGGAGCGCGTGTTCGAGA
TTCGTGTAAGTTTAGG
340 RPL10_56361 6134 RPL10 AGATTTATTAACGGGCGGGGCGGCGGTTTATGGCGATATTAGGATTTTTA
GTGGTTGTAACGAAAAAGAGAGAGTCGGAACGGAAGACGAGAATAGATT
341 RPRC1_56316 55700 RPRC1 TTGGGGTTATAGGCGCGTTATTACGTTCGGTTAATTTTTTGTATTTTTGGT AGAGTGGGGGTTTCGTTACGTTGTTTAGGTTGA
342 RPRC1_56318 55700 RPRC1 TTTTTACGGTTTTTACGGTGATCGGTTTCGTTTGGTTTTTTATTTTTAGATC GTAGTTTGTAGTTGAGCGTATGGGAGAGTAGTATCGT
343 RPRC1_56320 55700 RPRC1 GCGTTAGGTTGGTTTTTGTTCGGTTTTACGTGGGGTTTTTTTTAGGGTCG
TAGTTTTTATTTTGGTTTCGGTCGTTGCGGGGTAGTGTGATCGCGTTGCG
ATTTCGAGTAAGGAAGG
344 RPRC1_56335 55700 RPRC1 GAGGTTAGGTTTAGTTCGGGCGGTATAATAGGGTTGGGTCGAGGTCGG
GATTGGGTCGGCGTCGGGCGGGGAACGGGTTCGCGATCGTAGTCGAGA
GATTTCGG
345 RPRC1_56336 55700 RPRC1 TTTTTATTTAGTTTTTAGGCGTTGCGGCGCGTATTAAGACGCGGACGTAG
GGGGAGGTAGTTAGCGCGGCGAATTTTTGGAGTCGTGGAGTTCGTTCG
GT
346 RUNX3_1 1669 864 RUNX3 TTTTAGAGCGGGGTATGGTATCGAATAGTATTTTCGATTTTTTTTCGATTT
ATTCGTCGATTTTTATTCGCGGTGAGTAATAATAGTTTAG
347 RUNX3_2 864 RUNX3 TATTTATTTTGAAGGCGACGGGTAGCGTTTTGTTGTAGCGTTAGTGCGAG
GGTAGTACGGAGTAGAGGAAGTTGGGGTTGTCGGTGCGTACGAGTTCG
TTTGCGTGGTTCGTTA
348 RUNX3_23117 864 RUNX3 TTAT AGGATGCGAGAAGTTTGTTCGCGGTTTTGGTTTATTGGTTGGGTCG
CGGTTATTTGGGTCGTGATGTTACGGTTTTT
349 SALL4_12833 57167 SALL4 GAGGCGTAAGTAGGCGAAATTTTAGTATATTAATTCGGAGGAGGATTAGG GCGAGTAGTAGTCGTAGTAGTAGATTTCGGAGTTTGTAGATGCG
350 HIN1_1 92304 SCGB3A1 TAGGGAAGGGGGTACGGGTTTTTTAGGGTTCGTCGGTCGTAGTAGGAAG TTGGTTAGGGTACGGTCGTGAGCG
351 HIN1 3 92304 SCGB3A1 GAAGTTGGTTAGGGTACGGTCGTGAGCGGAGCGGGTAGGGTTTTTTTAG
GAGCGCGGGCGAGGTCGGCGTTGGAGGGGCGAGGATCGGGTATAAGA
AGTT
352 SCN1 B_56293 6324 SCN 1 B TTATTCGGAGAGGTCGGTGTAGCGGGCGTTTTTCGGGTCGTTGCGGTAA
AGGTTGGGCGGTCGCGTTTTTTTTTCGCGGTGATTTATTTCG
353 SCN1 B_56297 6324 SCN 1 B GGGTTTAGTTTCGGGGTTCGGCGTTCGGGTATTTTCGTCGCGCGGGCG
GCGGTCGTTGTTCGGGTTGTTGCGTTGGCGCGCGTAGTCGGGTATATTC
GTTA
354 SEMA3B_56291 7869 SEMA3B GTATCGTTCGTCGTGTCGTAAAGTTTAGAGGGCGGATCGGGTGATCGGG
TAGGTAGTCGGGATTAGTTGGAGACGGTAGTTAGGCGGGA
355 SESN1_29753 27244 SESN 1 TTTTAGATAGGGCGGGTTTTCGGGGAGTAGCGTTTCGGTTTTTTTGCGTT
TTTCGGGGGAAGGGGTTGTTTGCGGTGGGTGGGAGATTTGCGTATCGT
GAGTCGTTTTT
356 SESN1 29754 27244 SESN 1 ATTAGGACGAGGTATTTGGGCGAGGGGTTGAGCGGGTCGTTGGGTATTT
GGGCGGGTGGGTATTCGGTCGAGTTGTTTTGTGTAATACGGAGGTTACG
TTGTTTTT
357 SFN 2810 SFN TGGTAGTTTTTATGAAAGGCGTCGTGGAGAAGGGCGAGGAGTTTTTTTG
CGAAGAGCGAAATTTGTTTTTAGTAGTTTATAAGAACGTGGTGGGCGGTT
AGAGG
358 SFRP1 9378 6422 SFRP1 GGGAACGGTATTGATTGTTCGTAGTGGTTAGGTTTGGGTATCGTTGTTTT
TAGAGTTAATTTTGGTGTTGGGAGTGGAGTGAGGGTAGCGGATGTATTT
GTATTTTGTGAGC
359 SFRP1 9379 6422 SFRP1 ATGTTTATGTCGGTTTTGCGTTTTGTTTTTCGCGACGTCGGGGTTGTTTTC
GTCGTTTTTTCGCGCGCGTTTTGTCGTAAATTTTTAGGGATTTTCGGGGA
TAAAAGGCGTAGT
360 SLC22A3 56237 6581 SLC22A3 ATGGGCGTAGGAGGTTTTCGTACGCGTAAGGGTTAAGGGTTGGAGGGG
GTTTTTTCGGGTATAGGTATCGGATTCGGGGCGCGCGCGCGCGTTTTTA
GTTTTCGGGTTGTATTTCGGGTC 361 SLC22A3_56240 6581 SLC22A3 TTTTTCGGGTTTTAGTCGTAGCGTTCGGTTAGCGTCGCGGTATTTGGTTC GCGGTATTAGTAGTGGTCGGGTTGCGTGTTTAGGAAGATTACGT
362 SLC2A14_56224 144195 SLC2A14 TGAGTTTAGCGAGATTACGAGGTTATCGGAAGGAACGAACGATTTTTGAC GCGTTGTTTTAAGAGTTGCGATATTTACGGCGGAAGTTTGTAGTTTTAT
363 SLC35A5_56220 55032 SLC35A5 TAGTGTTACGTGAGGTTTCGGTGGCGGCGTAGTTACGGTAAGAGAGTGA
GAAGGAAGGGAAGTCGGAAGGGGCGCGAGTGAAGTAAAGCGAGGATAG
ATAGTTCGTAGAG
364 SLC35A5_56222 55032 SLC35A5 CGGATTCGATATTTATTGCGTAGGTACGTTTAGTGTTACGTTTTCGGAAG
GAGGGGAGAAGCGGTTTATTTCGTTTTAGTTCGGTTTTTTTTTTACGTTAC
GTGTCGGGAGAGTTTAGTG
365 SLIT1_23641 6585 SLIT1 TTTTCGTTATTAAGAGGTGTTTCGGCGTTAAAGCGATTAGCGTTGTTATTT
TTAGTTGGACGTTTGTAGTTAGCGTAGCG
366 SLIT1_23643 6585 SLIT1 CGTT AGGGTTGGGAACGATTAATGACGGTTAGTTATTTGAATAGATTAAT AATATATTAAACGGTTTAGGTTGAGGAGGGGCGGTCGAGTTATTTAATC
367 SLIT1_23651 6585 SLIT1 GCGTTATGGTGTTTTTATAGCGTTTCGTTCGCGAGTTAGACGGTAGTAGT
CGTTGATTATTTTCGTTCGGGGTCGTTTTTAGGTGTAGTTTCGGGGTAGA
GTTATCGAAGA
368 SLIT1_23653 6585 SLIT1 TTGTAGGCGGTTTGTAGTCGTTGAGTGGTCGTCGGGAGAGGGGGGTTG
CGGCGGGGGAGGGCGGGGAGGAGTTTGGTTTTGGATGTGTGTTTTTCG
ATTGATGGATGATTGTC
369 SLIT2_23672 9353 SLIT2 GAGGATCGGTTTAGGTTGCGGCGGAGTCGAGGGCGAGGGAGAGGTCG
CGTGAGTGAGTAGAGTTTAGAGTCGTGCGTTTTTAGAATTG
370 SLIT2_23676 9353 SLIT2 AGGGGAAGACGAAGAGCGTATATTTATAGTTTTTCGGTGTTGCGGGGGA TATTTTTGGGTACGTTGCGTAGCGTTAGTTCGTG
371 SLIT2_23678 9353 SLIT2 CGGTTTGTTTGGTTTTTAAGTTCGTTTTTAGTTAGTTTTTATTTAGTTTTCG
TAGTAGTTTTTATTTTTTATTTGGTTTTTTGGAGTTTTTCGTCGGAGTGTTG
ATTAGT
372 SLIT2_23681 9353 SLIT2 TAGCGGAGAGGAGATTACGCGTTTTTTGTTTTTTAAGGATGAATTTGGCG
GTAAAAGAGTTGGGGTTTTTAACGGTTGTTAAGATGTAGGGGTC
373 SOCS3_53602 9021 SOCS3 GCGTAGTTTTAGGAATCGGGGGGCGGGGCGCGGCGGTCGTTTATATATT
CGCGAGCGCGGTTTTCGCGGCGGTTTCGATTTGGATTTTTTGTTTCGTTG
TTGTCGTTTCGGT
374 SOCS3_53604 9021 SOCS3 GGAGGGGTTTCGTTTAGTTTCGGGAGTTTTTTTCGGTTTTTTTTTTTTTTTT
CGGGTTATTTTCGGTAGGGAGGTGACGAGGTAGGGGTAGAGCGGATGG
AAGTCGGAGATTTT
375 SOX9 29341 6662 SOX9 TGTAAGTGCGGGTATTGGTTCGCGAGTTTTTTGCGCGTCGTTTGCGTTTA
TATTATGAAGGCGTTTATGGGTCGTTTGACGTGCGGTTTGTTTTTGTTGG
AGTCGTTGACGCGTATCGGTATGGGTATTAGCGTTTAGTCGTAGTTTTTG
AGT
376 SOX9_56211 6662 SOX9 TGAATTGGTTATTTCGCGTTTTTTTAAGTGTTCGTCGCGGTAGTCGGTCG
ACGCGTTAGTTTTTTCGGGAGTCGTTTGTTTCGTATTCGGGTAGTCGAGG
377 SOX9_56213 6662 SOX9 GTAGTTAAAGGGCGGACGGTAGGGTGGGGGGGCGGGGGGTGTTGCGG
GGAAGGCGGGGGATTCGGGATTTTTAAGTGTGTAAGTTTGTCGTATTTTC
GGAATGTTAGA
378 SPFH2_55669 11160 SPFH2 GGACGAGTACGGAGCGTTTGTAGGGATAGTTTGGTACGCGGTTTTCGTT
TTTTCGTGCGCGCGTTGGGTTTAGTTGTCGTTTAGGGTCGGGGTTGATT
CGTTATTT
379 SPFH2_55675 11160 SPFH2 AGGTTTAACGTAATCGGGGCGCGGATATTTGCGAGGCGTTTATTTTATCG
TGGCGGTTTTCGGTTATCGATTTGTAGTTGCGTTTTT
380 SYK_53559 6850 SYK ATTCGGGTATTTTAGGGCGTTTAGGTTTTTAGGGAGCGCGGAAAGTGCG GTCGCGGTTCGGTTTTCGGGAGACGCGGGATTGGGATTAGGTATAGC
381 SYK 57767 6850 SYK GTATGAAAGTAGCGCGTCGTTTTTCGT AATTTTTTTTTTTTATTAGAAAGTT TCGGTCGGGTAGGGGACGCGGTTATATTTATTTGC
382 SYN E 1 23345 SYNE1 GTTGGGTTTTCGTAGTTTTGTAGATCGCGTTCGATTTCGGCGATAGGGC GGGCGGATAGTCGCGTATTTTCGGGGTTTCGTCGAGTTTGGGCGTAG
383 TAC1_56185 6863 TAC 1 ATTTGTAGACGGAAGTAGGTCGTTTCGGATTGGATGGCGAGATTTCGATT
TTTTTAAAATTGCGTTATTTAGAATTTAATTGGGTTTAGATGTTATGGGTAT
CGACGAGTTATCGTTTCG
384 TAC1_56187 6863 TAC1 GGGTATTTATTGCGACGGATAGTTTCGCGGGGTGTTGAGTTTTTTTGGTT
TTTTCGAGCGTACGTTGGTCGTTTCGTATTTTCGGTAGTTGTCGTCGG
385 TCL 1 A 8115 TCL1A GACGTTATGGTCGAGTGTTCGATATTCGGGGAGGTAGTTATCGATTATTC GGATCGTTTGTGGGTTTG
386 TCL1A_13087 8115 TCL1A CGATGGTTAAGGGTAGTTAGGCGTGTTGTTTTTCGTTTAAATATACGAATT
TTTTTTAGGTTTATAGGCGGTTCGGGTGGTCGGTGATTGTTTTTTCGAGT
GTCGGGTATTCG
387 TEX14_56176 56155 TEX14 TTGTGGGTTAGTTTTTAACGGTCGTTGGGAGGGATTAGTTCGTATTTTTC
GTATTTATTTACGTTTTTTCGGGAGTCGTATTTCG
388 TF 56164 7018 TF CGAGTTCGATTGTGTTCGTTGTTTAGCGTCGTATTCGGAAGATGAGGTTC GTCGTGGGAGTTTTGTTGGTTTGCGTCGTTTTGGGTGAGTGCGGGTACG GGGTAGTATCGTAGAGTCG 389 TF 56170 7018 TF TAGGTTTTTAGTTTGAGCGCGGAGTGTATGTAGGTTGCGCGGTGGTCGT TCGGGTTGTAGGGAACGCGCGGGTTAGCGATTTTGCGGTGTTATT
390 TFPI2 7980 TFPI2 GTTCGTTGGGTAAGGCGTTCGAGAAAGCGTTTGGCGGGAGGAGGTGCG CGGTTTTTTGTTTTAGGCGGTTCGGGTGTTCGTTTTATG
391 TFPI2_12620 7980 TFPI2 CGGGGTGATAGTTTTCGTGTATGAATTAGTTATTTTTTAGGTTTCGTTTCG
GCGGGGGTCGGTCGGACGTTCGTTTCGTATAAAGCGGGTATTCGGGTC
GTTTGGAGTAGAAAGTCG
392 THBS1_57460 7057 THBS1 CGAAAGTTTTTGCGTTATTTCGCGGGTTTTTTTAGGTGGTTTTTTTAGTTT
CGTTTTTTTTCGGGATGTTTGTTGATTATTTCGAGTTCGCGTGGCGTAAG
AGTACGAGCGTC
393 THBS1_57463 7057 THBS1 GGGCGATTTATTTGTGTGTATCGGAGCGCGGCGGTCGGGAGCGGTGGC
GAGGGCGGCGAGGGTTGGAGGGGCGCGGGGAATGTTTGTGCGTTCGG
AGTAGAGGTTGTTTT
394 THBS2_18771 7058 THBS2 GGATCGAGTTAGAAAGGCGTAGGTTTTGTTGGAGCGAGAGATCGGTTTT
GTAGTGTAGGATGTTTCGGTGGACGTGGTCGAGCGGTTTTCGGGTAGCG
CGGTTTTATAT
395 TIMP3 7078 TIMP3 GCGTCGGAGGTTAAGGTTGTTTCGTACGGTTCGGCGGGCGAGCGAGTT
CGGGTTGTAGTAGTTTCGTCGGCGGCGCGTACGGTAATTTTGGAGAG
396 TNFRSF11 B_30721 4982 TNFRSF11 B TATCGGGTTGAGGAATAAGGCGGTTGTTGTTTTCGCGGGGTTTTGAGGT TTTTCGGTTTTTTTTCGTTAGCGGTTAGTTTTT
397 TNFRSF11 B 30722 4982 TNFRSF11 B TGGTTTAGGGATTTATTACGAGCGCGTAGTATAGTAAGTTGTTTATTGTG
GTTTTCGGAAATTTTAGGGGTTTGGAGGCGGCGGTTGGGCGAGCGTTTC
GGTGCGTTTTCGTAGTT
398 TRPV2_18803 51393 TRPV2 TTATTTCGTAGGTTGAGGTTAGGGCGTGGCGGTTGTTGGGATTTCGGAG
TTTTTTAGTAGTAGGGGTTGCGGGAGGAAGTGAAGTCGGGAGGGGTTGT
CGGCGTTGATAGTAGAGGA
399 TSLC1 23705 IGSF4 GGTGAGTGACGGAAATTTGTAACGTTTGGTTCGTTAGGTTAGATGTATTC
GGTGTGCGGGAT AGAGGATTTTTTTAAGGGAGATTTTTTAGTCGTCGGTT
TGATATAGCGATTGT
400 TSLC1_1 23705 IGSF4 ATTGGTTTGTTCGGATTTCGTTTTTAGCGTATGTTATTAGTATTTTATTAGT
TGTTCGTTCGGGTTTCGGAGGTAGTTAACGTCGTTAGTTTGAGGTAGGT
GTTCGATATGGCGAGTGTAGTGTT
401 TSPYL6 388951 TSPYL6 TCGTTTGGAGTTAATCGTGTTTTTATCGTTTCGGTTTTTAGAGGAGGGGG
TCGCGTTTTAGGATTTCGTAGATGG
402 TSPYL6_12624 388951 TSPYL6 TTCGAGAAAAGAGTAAGGCGATAGAGGTAATGGTAGATATGTTTGATGGT CGTTTGGAGTTAATCGTGTTTTTATCGTTTCGGTTTTTAGAGGAG
403 TSPYL6_12626 388951 TSPYL6 TTATTCGTTATTTTGTGTGAGGAACGTTGACGTTATTT ATTTTTGGTTTGG
TTTTTATTTATTGTTGTTTTGGTCGTTATTAATATGAGTTTTTCGGAGAGTT
TTTATAGTTTCGT
404 TWIST1_3 7291 TWIST1 GTTAGGGTTCGGGGGCGTTGTTCGTACGTTTCGGCGGGGAAGGAAATC
GTTTCGCGTTCGTCGGAGGAAGGCGACGG
405 TWIST1_9329 7291 TWIST1 TTTAGTTCGTTAGTTTCGTCGGTCGACGATAGTTTGAGTAATAGCGAGGA
AGAGTTAGATCGGTAGTAGTCGTCGAGCGGTAAGCGCGGGGGACGTAA
GCGGCGTAGTAGTA
406 UCHL1_13107 7345 UCHL1 TAGGTTTTATAGTGCGTTTGGTCGGCGTTTTATAGTTGTAGTTTGGGCGG
TTTCGTTAGTTGTTTTTCGTTTTTTTTAGGTTATTTTTGTCGGGCGTTTCGC
GAAGATGTAGTTTAAGTCGATG
407 UCHL1_57523 7345 UCHL1 TTGTTAGTAGTCGGAATCGGTGGGCGTTTTCGTTTCGGCGTTCGCGGGG
GAGGGGGTTAGAGACGGCGCGAGACGTTATTTATTTTGTTT
408 WIF1_23405 11 197 WIF1 ATGTTTTAGAGTTAGAGCGCGGCGGTAGGGAAGGCGTTTTTTCGGGTTA TGTTGTTTAGGATTTTTTCGTTGTCGGGAAAATT
409 WIF1_9096 11 197 WIF1 GCGTCGTTAGATATTTTGTTGCGTTGTAGTTTTTTTAGTTAGGGTTGTTTT CGTTTAGACGGTTGGGCGCGTCGTTTTTCGGTTTGGGTGTTA
410 WT1_1 7490 WT1 TGTGTTATATCGGTTAGTTGAGAGCGCGTGTTGGGTTGAAGAGGAGGGT
GTTTTCGAGAGGGACGTTTTTTCGGATTCGTTTTTATTTTAGTTGCGAGG
GCGTTTTTAAGGAGTAGCG
411 WT1_2 7490 WT1 GGATATACGTGGAAGTCGGGTTTTGTAGTAAGAGGAAGTTTAGGATCGC
GGCGAGGAGACGGCGGGGTTCGGGCGTTTGGGTTGTCGTTTC
412 ZAP70_57514 7535 ZAP70 GCGGAGAGTGTGAGCGGTAGGGTTTTTTTTTTAGGTTGGGGGTTTGGGT TGTAGGGAAATTTAAATCGGTTTTGTAAGTTTCGATTTT
413 ZIC1_57510 7545 ZIC1 GGGCGGGTTAATGAGTTGCGCGCGGCGTTTCGGCGCGTTTTTCGTTGG
CGCGGCGGTTGAGGGCGGGGGGAATGCGGGCGTATTAATGGGCGTTA
GCGTCGGTAGTACGTGATATTTT
414 ZIC1_57513 7545 ZIC1 GTTAGGTAGATTTTGTCGGTTCGGTTCGTATTTTTTCGTCGTTTAGTTTTT
GTCGAGAGGGTCGCGCGTTAAGGTTGTTCGGGG
415 ZMYM5_57502 9205 ZMYM5 ATTTGGGGATTTCGGTTTCGGCGGTAGGGTGGTAGCGCGGTTTTGATAG
CGAAGATTCGGTCGGGGCGTTTTTTTTTCGTTTAATTCGTTTTTCGTTATT
ATGCGGTAGGAGAG
416 ZMYM5 57506 9205 ZMYM5 GGGTGTGGTTCGTATTTGACGGATAGTTTTTCGGTTATTTTATCGAGGGG
GCGTTTTGAAGGACGGTCGTCGGAGTTAATGGG 417 ZNF195_57489 7748 ZNF195 TTTTGATTCGGGGAGACGAGGGTTTGCGGGCGAAGAGTTGTCGGGGGG ATATGGTTTTGGGTTTGGGTTAAAGTCGTTTTGTAGGGATGCG
418 ZNF195_57492 7748 ZNF195 ATTTAGCGGGGTTTTTGTTTCGATAGAGTTTTATTTTTTGTTTTCGCGGTT TTGTGTTTTTTGTTAGTCGTAGGTCGTGTGATTCGTA
419 ZNF365_57483 22891 ZNF365 TAGTGTAATTTTGAGTCGGATCGGGTTTTCGGTTGGGTGGCGGGGGGGT
AGATGATGTTTTCGTTAGTTGGGTAGAGTAAGGGTGTTTTTCGCGGAGGT
GGTAGTT
420 ZNF365_57485 22891 ZNF365 TTTTTCGTTGCGTGTTAGTTTACGGGTGTTAGAGTGGATTCGTGTGTATA
TGAGTTTTTGTTGGTGGGGGTCGGGAAGTCGGTTTTT
421 ZNF432_57478 9668 ZNF432 TTTATGGAGAGCGGAATGCGGTTTTTCGGTTTTTTTTTAGGAGGAGACGC GTTCGCGTCGGCGTCGTTTCGTCGTTTTGGG
422 ZNF432_57479 9668 ZNF432 TTTAGTTTTTAAGAGGTCGCGGCGATTTTAAGGAGAGTTAGGTTAGTGTG
TGGGTTAGGGCGTGGAAACGTGGTGGGAGGGAGTTTTTTGAGGGCGTT
TGCGTTGTTGGTGGCGTTATAT
423 ZNF655 79027 ZNF655 TTATCGAGAAGCGTCGGTTTCGGGGTTGTTTATAGCGGTTCGGGAGAGG
TTGTGGTGGTTTCGAGCGCGAGTGTGTAGGTGATAGGATAGCGGTTAGG
TTCGTTTTTTTTTTCGGT
424 ZNF655_12792 79027 ZNF655 GTCGTAGTTATTGTTGGTATAACGGTTAGAGACGTTTAGTGAGTTAGTAT
GGAGGGTAGTGGGATCGGAAAAAGACGTGGAAAAGTTGCGA
EXAMPLES
Example 1
Materials and Methods
[79] Cell culture and treatment. MCF7, MDA-MB-231, MDA-MB-468, T-47D, HT-29, Caco- 2, Colo320. SW480, RKO, HCTl 16 cells and isogenic DNMTl/3b genetic knockout derivatives were maintained in culture as recommended by American Type Tissue Culture (ATCC). For drug treatments, log phase cells were cultured in the appropriate media (Invitrogen) containing 10% FBS and Ix penicillin/streptomycin with 5 μM 5aza- deoxycytidine (DAC) (Sigma; stock solution: 1 mM in PBS) for 96 hours, replacing media and DAC every 24 hours. Cell treatment with 300 nM Trichostatin A (Sigma; stock solution: 1.5 Mm dissolved in ethanol) was performed for 18 hours. Control cells underwent mock treatment in parallel with addition of equal volume of PBS or ethanol without drugs.
[80] Microarray analysis. Total RNA was harvested from log phase cells using the Qiagen RNEasy kit according to the manufacturer's instructions. Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturer's instructions. Hybridization was carried out according to the Agilent microarray protocol. Scanning was performed with the Agilent G2565BA microarray scanner.
[81] Data analysis. All arrays were subject to quality checks recommended by the manufacturer. All calculations were performed using the R statistical computing platform (Ihaka et ah, 1996) and packages from Bioconductor bioinformatics software project (Gentleman et ah, 2004). The log ratio of red signal to green signal was calculated after background subtraction and LoEss normalization as implemented in the Limma package from Bioconductor (Smyth et ah, 2003). Individual arrays were scaled to have the same inter-quartile range (75th percentile -25th percentile). Patient information, including clinical data and gene expression data, was obtained from and analyzed using Oncomine. Our analysis included microarray databases such as the Netherlands Cancer Institute breast cancer database (Liu et ah, 2007). The microarray meta-analysis algorithms and statistical analysis used was as previously described (Rhodes et ah, 2004). P-values were calculated using adjustment for multiple testing and false discovery as described (Rhodes e? α/., 2004).
[82] Methylation and gene expression analysis. RNA was isolated with TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. For reverse transcription-PCR (RT-PCR), 1 μg of total RNA was reverse transcribed by using Ready-To-GoTM You- Prime First-Strand Beads (Amersham Biosciences) with addition of random hexamers (0.2μg per reaction). Bisulfite modification of genomic DNA was carried out using the EZ DNA methylation Kit (Zymo Research). Selection of primers used for MSP and determinants for CpG island localization and designation was accomplished using MSPPrimer (Brandes et ah, 2007). MSP was performed as previously described (Herman et ah 1996). Primer sequences are listed in Table Ia, Figure 2 and Fig. 6b. Bisulfite sequencing and RT-PCR was preformed as previously described (Suzuki et ah, 2002). Gene expression quantitation was performed using RT-PCR and the ID software package (Kodak). Decreased expression was defined as expression that was not detectable with RT-PCR or decreased by 2/3 compared to expression levels in normal tissue measured using the ID software to quantitate bands.
[83] Human Tumor Analysis. Formalin-fixed, paraffin embedded tissues from primary breast (30) and colorectal cancers (20) were obtained from the archive of the Department of Pathology of the Johns Hopkins Hospital. Analysis of breast tumors was performed on 12 early stage and 12 late stage primary cancers. Approval was obtained by the Medical Ethical Committee of Johns Hopkins Hospital. DNA was isolated using the Puregene DNA isolation kit (Gentra Systems). MSP analysis was performed as described above. Example 2
[84] We used an expression microarray approach (Schuebel et al, 2007) to comprehensively analyze breast and colon cancers. We included analyses in the specific breast cancer lines where individual mutations were identified by Sjδblom et al The strategy utilized is depicted in Fig. Ia. The microarray screen identifies hypermethylated genes that are re- expressed following treatment with the DNA methyltransferase (DNMT) inhibitor 5- deoxyazacytidine (DAC) but not following treatment with the HDAC I/II inhibitor trichostatin A (TSA) alone. Following filtering of genes with no basal expression on the arrays and of genes without promoter CpG islands, this approach allows the identification of hypermethylated genes with an accuracy of 75% with a false negative rate of 9% (Schuebel et al, 2007). From analysis of 4 breast cancer cell lines (MCF7, MDA231, Mb468, T47D), plus 6 colorectal cancer (CRC) cell lines (SW480, RKO, HCTl 16, Caco2, Colo320, HT29), we identified 56 (out of 189 CAN genes) that met these criteria, with an approximately equal fraction originating from breast and colon lines. These 56 genes were common to both datasets and were studied further.
[85] Using methylation-specific PCR (MSP) and RT-PCR, the methylation and expression status of these 56 genes was determined in the 6 colorectal cancer cell lines and in the same 11 breast cancer lines used in the large-scale sequence analysis reported by Sjδblom et al (HCC38, HCC1954, HCC1008, HCCl 143, HCCl 187, HCC1395, HCC1937, HCC2218, HCC2157, Hs578T, and HCC1599). Of the 56 genes identified, some were not amenable to methylation analysis or were not methylated in cell lines. Thirty-six of the genes ("common target genes") were found to be hypermethylated in either the 11 breast cancer lines, the colon cancer cells, or both (Fig. Ib). RT-PCR analysis demonstrated markedly reduced or no expression accompanying this hypermethylated state.
[86] To confirm our MSP results, we analyzed the methylation status of selected genes using sequencing of bisulfite-treated genomic DNA from samples that were used in the MSP studies. In all cases, bisulfite sequencing confirmed the results obtained with MSP. For another control, we also studied the methylation and expression status of the genes in a derivative of HCTl 16 human colon cancer cells in which the DNMTl and DNMT3b DNA methyltransferases were homozygously deleted (DKO cells). These simultaneous deletions result in nearly complete lack of DNA methylation and the promoter DNA demethylation and re-expression of all known DNA hypermethylated genes examined in these cells (Rhee et α/.32000). In all cases, the loss or absence of methylation at the promoter regions of the 36 DNA hypermethylated loci in DKO cells was associated with expression of these genes. Importantly, in all cases, these 36 overlap genes were also found to be expressed in normal colon and breast tissue.
[87] Next, we determined whether the 36 common target genes were hypermethylated in primary colon and breast tumors. Eighteen of these genes were methylated in cell lines only but not in primary breast or colorectal tumors or were methylated in normal breast or colon tissue. Importantly, the other 18 genes showed cancer-specific methylation - being hypermethylated in primary tumors but not in normal colon or breast tissue (Fig. 3). Among these 18 genes, the frequency of methylation varied between breast and colon cancers. Some genes were methylated in breast but not colon cancers while others were methylated in tumors from colon but not breast. Most importantly, however, while only 6% of all CAN genes (12/189) are mutated in both colon and breast cancers, of the 18 CAN genes with cancer-specific methylation, 67% were hypermethylated in both colon and breast tumors (12/18) (Fig. 4). These results suggest that when epigenetic silencing is taken into consideration, the biological alterations of a significant number of genes in breast and colon cancers may share more similarities than apparent from mutational analysis alone. We investigated the number of genes (from among the 18 genes showing cancer-specific methylation) that are hypermethylated per tumor for both breast and colon cancers. Although most genes are methylated to some extent in both tumor types, a greater proportion of the genes are hypermethylated in colon cancer compared to breast cancer.
[88] We next analyzed the expression status of the 36 common target genes and compared this to the methylation state of each gene. Although hypermethylation was accompanied by loss of gene expression in nearly all cases, loss of gene expression in the overlap genes can occur by mechanisms other than methylation. Potential mechanisms include repressive chromatin modifications, mutational changes outside the coding regions that destabilize the mRNA, or coordinate downregulation of relevant pathways. For example, p21 expression is frequently decreased in tumors with inactivating p53 mutations (Matsushita et α/., 1996).
[89] We first determined whether cancer-specific methylation of the common target genes would correlate in any way with tumor stage or grade. We determined this, first, by directly analyzing the methylation state of breast cancers of varying stages (1-4) and grades (1-3). We found that SYNEl and COL7A1 are preferentially methylated in advanced tumors and PTPRD, SYNEl, and EVL are preferentially hypermethylated in high grade tumors (Fig. 5). For example, in stage 1 and 2 tumors, SYNEl is silenced 8% (1/12) of the time whereas in stage 3 and 4 tumors, the frequency of silencing is 50% (6/12). This is consistent with a role during tumor progression or during initiation of tumors pre-destined to evolve aggressive clinical behavior.
[90] Tumor stage and grade are strong prognostic determinants of disease-free survival and propensity for metastases in breast and colon cancer. Given our results above, we sought to validate whether expression of the genes we identified to be targets of hypermethylation and mutation affected clinical endpoints using data from external cohorts. Gene expression signatures from tumors have proven very useful for predicting clinical outcome (van de Vijver et ah, 2002; van 't Veer et ah, 2002). To begin to address this question, we analyzed an extensive microarray database, utilizing large numbers of expression profiles on very well documented clinical samples from published expression microarray studies. The microarray meta-analysis algorithms and statistical analysis used was as previously described (Rhodes et ah, 2004). These databases have been instrumental in a number of cancer gene discovery efforts (Lu et al, 2007; Fu et ah, 2006; Tomlins et ah, 2006).
[91] We first verified whether we could see in the databases the key predicted relationship between DNA methylation and repressed gene expression. Unlike for gene mutations, which alone could indicate either oncogenic or tumor suppressor changes, the occurrence of hypermethylation suggests the latter in genes targeted by both mechanisms. We, thus, asked whether genes undergoing a significant incidence of cancer-specific methylation correlated with decreased expression in tumor versus normal tissue. Genes undergoing cancer-specific methylation with low frequencies of methylation would not be predicted to have obvious gene expression correlations in the large database sets. We analyzed the following genes: COL7A1, PTPRD, GPNMB, APC2, ICAM5, EVL, SYNEl, and MMP2. As expected, all these genes were predicted by our analysis of microarray data to have decreased overall expression in breast and/or colon cancer compared to normal tissue (p-values 0.047 - 2.9E-7). These in silico results are consistent with the observations we made with direct laboratory analyses. [92] We next examined whether decreased expression of the genes undergoing cancer-specific silencing correlated with the key clinical characteristics. Decreased expression levels of 7 genes was associated with unfavorable clinical characteristics in either breast, colon cancer, or both. Importantly, these genes included the 5 genes - SYNE 1, COLlAl, PTPRD, SYNEl, and EVL - for which, in the studies described in Fig. 5, we found relationships between stage and grade in our studies of actual tumor samples. Importantly, decreased expression of 5 of the genes predicted for decreased disease-free or overall survival in these cancers (Fig. 5). These relationships are highlighted by the fact that, when we also analyzed several CAN genes which we directly determined to not have altered expression levels in breast or colon cancers (GGAl, PTPN14, ABCB8, OTOF, SIX4), the clinical endpoints we mentioned above were not associated with decreased expression of any of these genes.
[93] Related to the above correlation with survival, decreased expression of 5 of the genes was seen in metastases when compared to primary tumors, such as GPNMB, LGR6 and, especially, PTPRD. Intriguingly, GPNMB encodes the glycoprotein nonmetastatic melanoma protein B, which has been shown to be differentially expressed between highly and lowly metastatic melanoma cell lines and xenografts such that markedly lower expression levels characterizes metastatic cells and overexpression of the GPNMB protein lowers metastatic potential (Weterman et ah, 1995).
[94] Finally, four genes are underexpressed with increasing tumor grade (COL7A1, SYNEl, PTRD, EVL). Since grade is a strong predictor of local recurrence and metastasis, silencing of these genes may be clinically relevant determinants of prognosis (reviewed in Soerjomataram et ah, 1995). It is important to note that our direct analysis of tumor samples is consistent with our analysis of microarray gene expression data from these other cohorts.
Example 3
Strategy to identify supplementary gene targets of hypermethylation in breast cancer.
[95] The re-expression information of four cell lines (ATCC, P.O. Box 1549 Manassas, VA 20108 USA) isolated from pleural effusion of patients with breast cancer was used to elect initial gene candidates for hypermethylation. The cell lines concerned: 1. MCF7: adenocarcinoma, derived from non-metastatic breast cancer
2. T-47D: ductal carcinoma, derived from non-metastatic breast cancer
3. MDA-MB-231 : adenocarcinoma, derived from metastatic breast cancer
4. MDA-MB-468: adenocarcinoma, derived from metastatic breast cancer
[96] Cell culture, microarray and data analysis was done as described in Schuebel et al, 2007. Initial candidate genes were selected based on the following criteria:
a. The gene had to be re-expressed under AZA treatment. The re-expressed genes were ranked as a top tier gene or a next tier gene. A gene was termed as a top tier gene if the expression is up regulated by more than two-fold in the AZA treated versus mock sample on the Agilent whole human genome expression microarray platform; if it showed an enrichment between 1.4— fold and two- fold it was termed as a next tier gene b. The gene had to be silent, i.e. having no basal expression, in the mock cells c. The gene should not respond to TSA treatment alone.
[97] Different strategies were taken towards identification of good candidate genes susceptible to hypermethylation (computational and verification strategies based on cell lines and primary tumors:
1. As shown by Schuebel et al. fSchuebel et α/.2007) and based on the sequencing project from Sjδblom et al. (Sjδblom et al, 2006), promoter CpG island methylation and subsequent gene silencing of genes known to be mutated in cancer is more frequent than the mutations themselves. Therefore the mutated genes identified by Sjδblom et al. (Sjόblom et al, 2006) in either colon or breast cancers, were used to fine-tune the candidate methylation genes ranked as top or next tiers. Taking into account the re-expression data from all 4 cell lines, 31 genes were found to adhere to this category. Eleven of these 31 genes showed tumor specific methylation in breast cancer and were selected for further analysis.
2. Another 89 genes were selected from the top and next tier genes from the breast cell lines based on their overlap between or specificity for certain cell lines (e.g. genes in the top tier of both metastatic cell lines, but not in any of the non- metastatic cell lines), expert knowledge on the function, history and mutation status in other cancer types, of which 41 showed tumor specific methylation in primaries and 48 were methylated in the cell lines.
3. The promoters of all the selected and clearly annotated top and next tier genes were separately mapped on a genome-wide alignment of all promoter associated CpG islands. The genes were selected if they were located less than or equal to 9 ancestral nodes from an established list of 56 markers (see BROAD analysis hereafter). These genes were overlapped with a list of polycomb marked genes in either human embryonic fibroblast (Bracken et ah, 2006) or human embryonic stem cells (Lee et ah, 2006), in order to have a more concise list of cancer specific methylation events. Using this approach, 211 (17 overlapping with strategy 1 and 2; 194 previously unseen) genes were identified. The genes were ranked according to the number of known methylation markers and the number of ancestral nodes between the gene and the known methylation marker.
BROAD analysis: Genome-wide Promoter Alignment
[98] The "Database of Transcription Start Sites" (DBTSS) (Suzuki et al.,2004; Suzuki et al.,2002) mapped each transcript sequence on the human draft genome sequence to identify its transcriptional start site, providing more detailed information on distribution patterns of transcriptional start sites and adjacent regulatory regions. From -14,500 well- characterized human genes present in the Affymetrix GeneChip Human Genome U133A Arrays, 8793 sequences were extracted from the DBTSS (Suzuki et al.,2004; Suzuki et al.,2002) (DBTSS, version 3.0 based on human assembly build 31). The remaining genes (14,500-8793=5707) on the Affymetrix array contained no reported transcriptional start site (TSS) according to DBTSS. All the promoter sequences were subsequently aligned by ClustalW algorithm (Li et ah, 2003; Thompson et ah, 1994). Treelllustrator (Trooskens et ah, 2005) was used to visualize the large guide tree in addition to indicating the location of the known markers. Some regions on the "circle" are denser in known markers than others, indicating that there might be a sequence mechanism located in the small region around the TSS which makes certain genes more methylation-prone.
[99] A total number of 294 genes comprising the 263 candidate genes identified by the aforementioned 3 strategies and known published cancer markers, were retained for further analysis. Duplicates, imprinted genes and genes for which primer design was not possible were excluded from the list. The candidate genes were further tested on differential methylation using the OpenArray™ plate real-time qPCR system (BioTrove Inc.)
Example 4
Assays for identifying differential methγlation in breast cancer.
[100] An openArray™ plate real-time qPCR system (BioTrove Inc.) is applied on samples from breast cancer tissue and samples isolated from (corresponding) normal breast tissue. From each sample, up to lμg of genomic DNA is converted using a bisulphite based protocol (EZ DNA Methylation Kit™, ZYMO Research, ORANGE, CA.). After conversion and purification the starting material is applied per sub-array of an OpenArray™ plate on the real-time qPCR system offered by BioTrove Inc. using the DNA double strand specific dye SYBRgreen for signal detection.
Assay details
[101] If possible, multiple assays were designed for each of the genes to be investigated. Assays for one and the same gene differed in the primers used for assessing the methylation status of the concerned gene. For instance, to assess the methylation status of the ABHD3 gene, assay ABHD3 11682 uses the sense primer GATATTCGTCGTAGAGCGGAA (SEQ ID NO: 1) and the antisense primer CAAAAATAAACCGAAAACTAACGA (SEQ ID NO: 425) whereas assay ABHD3_55542 uses a different primer set consisting of the sense primer GGTTTTGCGTTATTTTCGGT (SEQ ID NO: 2) and the antisense primer CTTATCCTAAACTTCAACGTCGC (SEQ ID NO: 426). The different assays and their corresponding primers and converted amplicons for 150 different genes (out of the 294 candidate genes) and a number of published markers are detailed in Table IA, Table IB, Fig. 2 and Fig.όB. Primer design
[102] An example on primer design spanning a large region of the promoter is provided in Fig. 6A and 6B for PPP1R13B. Primer design is limited to the promoter region. For the assay SLC2A14-JHI, the TSS is positioned at 7907318 and the assay at 7916993, positioning the assay at - 9675 bp relative to the TSS. For the assay NEFH-JHI, a TSS is positioned at 28196907 and at 28215181 whereas the assay is positioned at 28206218, resulting in a region of - 9675 bp and 8963 bp relative to the TSS respectively. For the GPNMB-Bay assay, the TSS positions at 23252841, whereas the assay is positioned at 23253861 resulting in -1020 bp relative to the TSS.
Example 5
Breast marker ranking.
[103] The above identified candidate genes (Table 1: 424 different assays in total) were further tested on paraffin embedded breast biopsy material using the openArray™ plate real-time qPCR system (BioTrove Inc.). Differential methylation was assessed using the Biotrove platform as follows: DNA was extracted from FFPE breast samples, bisulphite converted, and selected regions of the particular genes were amplified using the primers as detailed in Table IA. Amplification was monitored in real-time set up using SYBRgreen. Data analyses designed to cope with inherent variance (i.e., noise) in measured Ct and Tm values were applied to withhold the top 60 best scoring assays for further testing on the Base5 methylation profiling platform (Straub et al. 2007).
Sample specimen
[104] A total of 196 paraffin embedded breast resection samples (97 breast cancer samples and 99 normal tissues) were used to find markers which distinguish cancer from non-cancer tissue based on methylation status. Normal tissue derived from breast reductions.
DNA extraction and Bisulphite modification
[105] DNA was isolated using proteinase K digestion and phenol/chloroform extraction. DNA concentration was measured using NanoDrop Spectrophotometer. From each sample, up to 2 μg of genomic DNA was converted using a bisulphite based protocol (EZ DNA Methylation Kit™, ZYMO Research, Orange, CA.). Detection of hypermethylation
[106] Methylation specific PCR (MSP) primers were designed for each of the genes assessed for hypermethylation (see Table IA). For some genes more primer pairs were designed giving a total of 424 different assays. These assays were applied on 8 sub-arrays of 2 OpenArray™ plates by BioTrove Inc. The beta-actin assay was applied on each sub-array as an internal control. Quality control was performed using an in vitro methylated DNA sample (Chemicon International, CA, USA; Cat.# S7821) and a negative control sample. The selectivity and the reproducibility were checked.
[107] After DNA conversion and purification, beta-actin copy number was determined by qMSP. The equivalent of 750 beta-actin copies per sample was applied per sub-array of an OpenArray™ plate on a real-time qPCR system (BioTrove Inc.) using the DNA double strand-specific dye SYBRgreen for signal detection.
[108] The cycling conditions were: 90° C-10 seconds, (43° C 18 seconds, 49° C 60 seconds, 77° C 22 seconds, 72° C 70 seconds, 95° C 28 seconds) for 40 cycles, 70° C for 200 seconds, 45° C for 5 seconds. A melting curve was generated in a temperature range between 45° C and 94° C.
Analysis of methylation
[109] For each combination of assays and samples two parameters were collected using an algorithm which is part of the standard data analysis package offered by the supplier. The parameters were the Ct value (threshold cycle number) and the melting temperature of the assessed amplicon.
[110] The following data analysis workflow was applied to the results created by the software which came with the system OpenArray™ system: Data was collected for each combination of assays and samples in the two sets of samples used. Results were filtered using the following approach. Read outs from not loaded reaction spaces were removed from analysis. Technical Control assays were removed from the data set. Assays known to not work for other than biological reasons were removed from the analysis. Per sub- array, signals were only interpreted if there was a positive beta-Actin call. Ct values > 0 for each gene were normalized using the Ct values collected for the gene beta-Actin. This resulted in two files containing the results for each set of sample. 201 samples were tested of which 6 gave invalid results. In total 82,712 reactions were performed of which 79,975 were valid. For the data analysis, 2 boundaries were defined: an upper bound on Ct and a lower bound on Melting Temperature (Tm). Samples below the banCt boundary and above the Tm boundary are considered to be "methylated", others (including all samples with no signal, i.e. Ct>40) are classified as "unmethylated". In both dimensions the set of candidate boundaries consists of all values in between 2 measurements, plus infinity (the equivalent of no boundary). The set of candidate models for "methylated" then consists of all combinations of candidate Tm lower bound and a banCt upperbound. A score is computed for each of these candidate models, as follows. Count: cancers inside boundaries = true positives (TP), cancers outside boundaries = false negatives (FN), normals inside boundaries = false positives (FP), normals outside boundaries = true negatives (TN). A binomial test was applied to find out how unusual it is to have at least TP successes in (TP+FP) trials where the probability of success is (TP+FN). The lower this probability value, the better. Then quality control data were taken into account to determine the most robust boundaries. Using the standard deviations (StDevQC) observed in the QC, a series of increasingly "noisy" datasets were generated. The measurements are replaced by a value randomly selected from a normal distribution with average equal to the observed measurement and standard deviation equal to StDevQC multiplied by a value that gradually (10 noise levels) increases from 0 to 2. Each time the score of the candidate model is computed by applying the 2 steps above (i.e., count and binomial test). All these scores (11 in total: 1 for "no noise" and 10 for noise levels 0.2,0.4,... ,2) are added up to obtain the ultimate accumulated score. The candidate model with the best (i.e. lowest) accumulated score is retained. This same score of the best candidate model for each marker is also used for ranking the markers.
Results ] A high throughput, real-time methylation specific detection platform was applied on two groups of samples isolated from breast cancer tissue and from normal breast tissue derived from breast reductions. A ranking method based on inherent variance (i.e., noise) in measured Ct and Tm values retained the 60 best scoring assays for further testing on the Base5 methylation profiling platform. This final selection of gene assays is detailed in Table 2.
Table 2: Top 60 breast marker ranking
Figure imgf000070_0001
Figure imgf000071_0001
Example 6
Selection of best performing assays: Base5 methγlation profiling platform
[112] The highest ranked assays resulting from above set out scoring strategy combined with expert selected assays were tested on tissue using the Base5 methylation profiling platform (Straub et al. 2007). These included following markers: ACTN2, APC2, BHMT2, CCK, CDOl, CKM, COL7A1, CST6, EREG, EVL, EYA4, FAS, FBLNl, FLNC, FOXL2, GDA, GPNMB, GPX7, GREMl, HEYL, HOXA4, ICAMl, KL, LY6K, MMP2, NEFH, NEFL, NEFM/NEF3, PLAU, PTPRD, SLC2A14/SLC2A3, STARD8, SYNEl, TCERGlL, TF, UCHLl, NPTX2, ADAM23, ADAMTS18, ATP2A2, HOXDl, RASSFlA, TACl, NDRG2, Beta-Actin, BRCAl, COX7A1, CSPG2, DSC3, GPXl, GSTPl/GST-Pi, HOXAlO, HOXA5, HOXA9, LEPR, MSH4, NEURL, CDKN2A/P16, RARRESl, RARRES2, RUNX3, S0CS3 and SYK.
[113] Primer and amplicon sequences for the majority of the genes are indicated in Table 1. Additional assay designs and details on expert selected assays are summarized in Table 3 and 4 below:
Table 3 : MSP Primer sequences
Figure imgf000072_0001
Table 4: MSP converted amplicon sequences
Figure imgf000072_0002
Sample specimen ] A total of 137 breast tissue samples (84 breast cancer samples and 53 normal tissues) were collected from various clinics. Normal tissue derived from breast reductions. The sample set selected for the Lightcycler analysis was also previously used in the Biotrove analysis in order to make a compared analysis.
DNA extraction and Bisulphite modification
[115] DNA was isolated using standard phenol/chloroform extraction. DNA concentration was measured using NanoDrop Spectrophotometer. From each sample, up to 1.5 μg of genomic DNA was converted using a bisulphite based protocol (EZ DNA Methylation Kit™, ZYMO Research, Orange, CA.).
Amplification and analysis
[116] Amplification by methylation-specific PCR (MSP) was performed using the Roche LightCycler LC480 in 384 well plate format with SybrGreen detection using following conditions: initial denaturation (activation) at 95° C for 10 minutes followed by 45 cycles (amplification) of 10 sec denaturation at 95° C, 30 sec annealing at 60° C and 1 sec elongation at 72° C. Melting curves are generated from 45° C until 95° C.
[117] An equivalent of 20 ng of genomic DNA was used per reaction. Cell lines were included in each run as positive and negative controls: CpGenomeTM Universal Methylated/Unmethylated DNA (Chemicon International, CA, USA; Cat.# S7821 and Cat.# S7822).
[118] Obtained amplicons were analysed using capillary electrophoresis on the Caliper LC90. Final methylation calling is performed using an in-house developed algorithm based on Ct-, Tm-, and amplicon length.
Results
[119] DNA methylation calls were compared between breast cancer and control patients. An assay ranking with above set of samples was generated and individual performance of the 18 best performing markers is summarized in Table 5. A one-tailed Fisher's exact test was used as a scoring function to rank the candidate markers. The calculation of Fisher's exact test was based on a formula as described by Haseeb Ahmad Khan in "A visual basic software for computing Fisher's exact probability" (Journal of Statistical Software, vol. 08, issue i21, 2003).
Table 5: Individual assay performance of breast markers
Figure imgf000074_0001
Example 7
Real-time MSP assay for detection of Breast Cancer
Marker identification and selection
[120] 10 assays were selected based on the highest ranking on Base 5 platform and on complementarity analysis: NDRG2_56603, GREM1_29777, TAC1_56187, H0XDl(2), SALL4J2833, CDO1_55928, LTB4R_31250, CDO1_55929, RASSFlA and NPTX2_57779. For these assays, analyte quantifications were performed by real-time MSP. These consisted of parallel amplification/quantification processes using specific primers and probes for each analyte and Molecular Beacon® assay formats on an ABI Prism® 7900HT instrument (Applied Biosystems).
Sample specimen
[121] A total of 196 paraffin embedded breast biopsy samples (100 breast cancer samples and 94 normal tissues) were used to find markers which distinguish cancer from non-cancer tissue based on methylation status. Normal tissue derived from breast reductions (most of the samples match to sample set from example 5).
DNA extraction and Bisulphite modification
[122] DNA isolation and bisulphite modification procedures are identical to example 5. To have enough material available for testing, the converted DNA was diluted with Tris-HCl ImM pH 8 adding up to ± 70μl of sample volume.
Amplification and analysis
[123] Real-time MSP was applied on a 7900HT fast real-time PCR system (Applied Biosystems). 5 μl of diluted modified DNA was added to a PCR mix (total volume 10 μl) containing buffer (16.6mM (NH4)2SO4, 67 mM Tris (pH 8.8), 6.7 mM MgCl2, 10 mM β- mercaptoethanol), dNTPs (5 mM), forward primer (6 ng/μl), reverse primer (18 ng/μl), molecular beacon (0.16 μM), and Jumpstart DNA Taq polymerase (0.04 units/μl; Sigma Aldrich). The primer sequences and molecular beacon sequences used for each of the genes are summarized in Table IA and Table 6 respectively. Cycle program used was as follows: 5 minutes 95° C, followed by 45 cycles of 30 seconds 95° C, 30 seconds 57° C (=plateau data-collection), and 30 seconds 72° C. In addition to the breast test genes, the independent reference gene β-actin (ACTB) was also measured:
ACTB sense primer 5' - TAGGGAGTATATAGGTTGGGGAAGTT - 3' (SEQ ID NO: 1482) ACTB anti-sense primer 5' - AACACACAATAACAAACACAAATTCAC - 3' (SEQ ID NO: 1483)
ACTB molecular beacon 5'-FAM-
CGACTGCGTGTGGGGTGGTGATGGAGGAGGTTTAGGCAGTCG-3'-DABCYL (SEQ ID NO: 1484)
Table 6: Beacon sequence details selected breast marker assays
Figure imgf000075_0001
Figure imgf000076_0001
[124] The results were generated using the SDS 2.2.2 software (Applied Biosystems), exported as Ct values (cycle number at which the amplification curves cross the threshold value, set automatically by the software), and then used to calculate copy numbers based on a linear regression of the values plotted on a standard curve of 8 - 8 x 10Λ5 gene copy equivalents, using plasmid DNA or PCR product material containing the bisulphite modified sequence of interest. IvM DNA and IvUn DNA (Chemicon International, CA, USA; Cat.# S7821 and Cat.# S7822) were included in each run as positive and negative controls. A run was considered valid when the following five criteria were met: a) slopes of both standard curves above -4 (PCR efficiency > 77.8%); b) rΛ2 of at least 4 relevant data points above 0.990; c) routinely included NTC not amplified; d) 10% of a lμg conversion reaction of IvM DNA (positive control) was detectable; and e) 10% of a lμg conversion reaction of IvUn DNA (negative control) was not detected within the standard curve.
[125] The ratios between the breast test genes and ACTB were calculated to generate the test result. Technical test run validation criteria used to classify samples as methylated, non- methylated or invalid are described in Table 7.
Table 7: Criteria to interpret the Real-time MSP data
(*) subjected to change based on larger clinical trials
(§) If the Ct value for the M-gene is > 40, then the calculated ratio is only theoretical and the sample is considered as non-methylated
Figure imgf000076_0002
Results
[126] The individual performance (% sensitivity and % specificity) of the 9 gene assays are detailed in Table 8 below (there was not enough sample material available to test the 10th assay: RASSFlA). Obtained specificity values ranged from 90 to 96%, corresponding sensitivity values from 37 to 91%. When sensitivity values are split up according to cancer stage, slightly higher values were observed for advanced stage compared to early stage cancers except for GREMl and SALL4, where a significant higher sensitivity was observed for early stage cancers. Samples were classified as methylated, non-methylated, or invalid based on the validation criteria set out in Table 7. In this linear interpretation model a sample is scored positive if the marker gives a copy number value above the cutoff. Cutoffs were adapted to obtain at least 90% specificity; leading to a high cutoff for NDRG2 (125), SALL4 (275) and LTB4R (250) (displayed in column 2 of Table 8). Cutoff of the reference gene ACTB was set at 200 copies.
Table 8: Specificity and sensitivity performance
Figure imgf000077_0001
Example 8
Methylation analysis SLC2A14, NEFH and TF
Sample specimen
[127] Primary paraffin embedded tissue samples used for this study were obtained from the archives of the Department of Pathology, Johns Hopkins University School of Medicine and the Department of Pathology, University Regensburg, with approval by the corresponding Institutional Review Boards. This study compiles data from FFPE primary breast cancers of tumor stages (TNM Stages) 0(DCIS)-4 (n=30; n=6 TNM SO/DCIS, n=6
TNM Sl, n=6 TNM S2, n=6 TNM S3, n=6 TNM S4).
[128] DNA of normal human breast samples for conventional MSP was purchased from Stratagene (n=6).
Methylation analysis
[129] DNA was extracted following a standard phenol-chloroform extraction method and bisulfite modified using the EZ DNA methylation Kit™ (Zymo Research). Primer sequences were designed using MSPPrimer Chttp ://www. mspprimer . or g) and are detailed in Table 9. Corresponding amplicon sequences are listed in Table 10. Methylation-specific PCR was performed as described by Herman et al, 1996.
[130] In Vitro methylated DNA (IVD) was used as a positive control for MSP. IVD was created by treating cell line DNA with Sssl methylase (New England Biolabs) as directed. DKO cell line DNA (DNMT double knockout) was used as negative control.
Table 9: MSP assay and primer details:
Figure imgf000078_0001
Table 10: Amplicon details (converted sequences issuing from the methylated version of the DNA )
Figure imgf000078_0002
Gel electrophoresis
11 [131] Amplification products (7.5μl of 25μl total MSP volume) were separated using electrophoresis on 2% agarose gels containing GelStar™ Nucleic Acid Gel Stain (Cambrex Bio Science) and visualized by ultraviolet illumination.
Results
[132] Based on the gel image and the use of M and corresponding U primer sets, the methylation status for each gene was assessed. Results for SLC2A14, NEFH and TF are shown in Figure 7. Methylation frequencies of 74%, 59% and 50% were obtained respectively. Positive and negative cell line controls, run simultaneously with the samples gave expected results. All 3 genes were completely unmethylated in the 6 normal breast controls tested.
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Claims

WE CLAIM:
1. A method for detecting breast cancer or its precursor, or predisposition to breast cancer, comprising: detecting in a test sample containing breast cells or nucleic acids from breast cells, epigenetic modification of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; P0U2AF1; PPP1R13B; PPPl Rl 4A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655; identifying the test sample as containing cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia, or as containing nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
2. The method of claim 1, wherein epigenetic modification of at least two genes is detected.
3. The method of claim 1 wherein epigenetic modification is detected by detecting methylation of a CpG dinucleotide motif in a promoter or a region surrounding the TSS of the gene.
4. The method of claim 3 wherein methylation is determined using methylation specific PCR, or an equivalent amplification technique.
5. The method of claim 3 wherein methylation is determined using bisulfite sequencing
6. The method of claim 4 wherein the methylation specific PCR or equivalent amplification technique is carried out in real time.
7. The method of claim 4 wherein the amplification employs hairpin primers (Amplifluor™), hairpin probes (Molecular Beacons™), hydrolytic probes (Taqman™), FRET probe pairs (Lightcycler™), primers incorporating a hairpin probe (Scorpion™), primers incorporating complementary sequences of DNAzymes that cleave a reporter substrate included in the reaction mixture (DzyNA™) or fluorescent dyes.
8. The method of claim 1 wherein the step of detecting utilizes at least one primer set selected from primers consisting of the nucleotide sequences set forth in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
9. The method of claim 1 wherein the step of detecting employs amplification of at least a portion of the at least one gene, and further employs at least one oligonucleotide probe which hybridizes to an amplicon selected from the group consisting of Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
10. The method of claim 9 wherein the probe comprises, consists essentially of or consists of sequences represented by Table 6 (SEQ ID NO: 1485-1494).
11. The method of claim 1 wherein epigenetic modification is detected by detecting diminished expression of mRNA of the gene.
12. A method for categorizing or predicting clinical outcome of a breast cancer, comprising: detecting in a test sample containing breast cancer cells or nucleic acids from breast cancer cells, epigenetic modification of at least one gene selected from the group consisting of GPNMB, COL7A1, PTPRD, SYNEl, and LGR6; providing a prediction of decreased disease- free or overall survival, higher stage cancer, or higher grade cancer if epigenetic modification is detected.
13. The method of claim 12 wherein higher stage or grade is predicted and the gene is selected from the group consisting of COL7A1, PTPRD, and SYNEl.
14. The method of claim 1 further comprising the step of prescribing a more aggressive therapeutic regimen if epigenetic silencing is determined.
15. The method of claim 1 further comprising the steps of prescribing more frequent diagnostic events if epigenetic silencing is determined.
16. A method of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetically silenced transcription of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; PON2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655; comprising: restoring transcription of said gene by administering one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, a histone deacetylase (HDAC) inhibitor, and a polypeptide encoded by said gene .
17. A method of treating a cancer patient comprising the steps of: detecting in a test sample containing breast cells or nucleic acids from breast cells, epigenetic silencing of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NA ALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF 195; ZNF365; ZNF432; and ZNF655;
adminstering a therapeutic agent that restores or increases expression of the gene.
18. A kit for assessing breast cancer or its precursor, or predisposition to breast cancer in a test sample containing breast cells or nucleic acids from breast cells, said kit comprising in a package: a reagent that (a) modifies methylated cytosine residues but not non-methylated cytosine residues, or that (b) modifies non-methylated cytosine residues but not methylated cytosine residues; and at least one pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; COL7A1; COL9A3; C0X7A1; CRIPl; CSPG2; CST6; CTAGlA; CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27; DDX43; DNAJA4; DUSP6; EIF5B; EPB41L3; ERCC3; EREG; ESRl; ESR2; EVL; EYA4; FAM20B; FAM84A; FAS; FBLNl; FBLN2; FBN2; FKBP4; FLJ21511; FLNC; FOS; GADD45G; GALE; GDFlO; GJB2; GPXl; GPX7; GSTPl; HCP5; HEYL; HHIP; HIST1H3G; HMG20B; HORMADl; HOXAlO; H0XA5; HUSlB; ICAMl; ICAM5; ID4; IGSF4; IL17RD; INGl; INHBB; IRF7; ITIH5; KCNG3; KIFlA; KL; KLF4; KRT14; KRTCAP3; LEPR; LGR6; LHX6; LIPG; LOX; LSMDl; LY6K; LZTFLl; MAGEHl; MAL; MET; MGC33846; MMP2; MSH4; MYCLl; NAALAD2; NAGS; NDP; NEDD4L; NEF3; NEFL; NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA; PLAGLl; PLAU; PLXNA4B; PNMA3; P0N2; POU2AF1; PPP1R13B; PPP1R14A; PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1; RP11-450P7.3; RPLlO; RPRCl; RUNX3; SCGB3A1; SCNlB; SEMA3B; SESNl; SFN; SFRPl; SLC22A3; SLC35A5; SLITl; SLIT2; S0CS3; SOX9; SPFH2; STARD8; SYK; SYNEl; TCLlA; TEX14; TFPI2; THBSl; THBS2; TIMP3; TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl; WTl; ZAP70; ZICl; ZMYM5; ZNF195; ZNF365; ZNF432; and ZNF655; wherein the region is within about 10 kb of said gene's transcription start site.
19. The kit of claim 18 wherein the at least one pair of primers is selected from Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495- 1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
20. The kit of claim 18 wherein the at least one pair of oligonucleotide primers amplifies an amplicon selected from Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471- 1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
21. The kit of claim 18 further comprising at least one oligonucleotide probe which hybridizes to an amplicon selected from the group consisting of Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
22. The kit of claim 21 wherein the oligonucleotide probe is selected from the group consisting of Table 6 (SEQ ID NO: 1485-1494).
23. The kit of claim 18 or 22 further comprising a DNA polymerase for amplifying DNA.
24. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-1503.
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