WO2005038046A1 - Procede de diagnostic base sur la methylation - Google Patents

Procede de diagnostic base sur la methylation Download PDF

Info

Publication number
WO2005038046A1
WO2005038046A1 PCT/AU2004/001398 AU2004001398W WO2005038046A1 WO 2005038046 A1 WO2005038046 A1 WO 2005038046A1 AU 2004001398 W AU2004001398 W AU 2004001398W WO 2005038046 A1 WO2005038046 A1 WO 2005038046A1
Authority
WO
WIPO (PCT)
Prior art keywords
methylation
msp
methylated
gene
round
Prior art date
Application number
PCT/AU2004/001398
Other languages
English (en)
Inventor
Anthony John Bais
Gabriel Kremmidiotis
Original Assignee
Bionomics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2003905593A external-priority patent/AU2003905593A0/en
Application filed by Bionomics Limited filed Critical Bionomics Limited
Publication of WO2005038046A1 publication Critical patent/WO2005038046A1/fr

Links

Classifications

    • 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
    • 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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention is concerned with a methylation-based diagnostic method.
  • CpG island regions comprise about 1% of vertebrate genomes and account for about 15% of the total number of CpG dinucleotides (Bird, 1986; Cross and Bird, 1995) .
  • CpG islands are typically between 0.2 to about 1 kb in length and are located upstream of many housekeeping and tissue-specific genes, but may also extend into transcriptional coding regions (Gardiner-Garden and Frommer, 1987) . Methylation of cytosine residues contained within CpG islands of certain genes has been inversely correlated with gene activity. Therefore, it is the methylation of cytosine residues within CpG islands in somatic tissues, which is believed to affect gene function by altering transcription (Cedar, 1988) .
  • An alteration in gene transcription may be mediated by a variety of mechanisms including disruption or remodeling of local chromatin structure, inhibition of transcription factor-DNA binding, or by recruitment of proteins which interact specifically with methylated sequences indirectly preventing transcription factor binding (Jaenisch and Bird, 2003) .
  • Several studies have shown that aberrant methylation of normally unmethylated CpG islands is a frequent event in immortalized and transformed cells which is routinely characterized by genome-wide hypomethylation and regional CpG hypermethylation (Jones and Baylin, 2002; Jaenisch and Bird, 2003) .
  • Hypomethylation promotes tumorigenesis by inducing loss of heterozygosity, mutation and possibly oncogene activation (Gupta et al . , 2003; Eden et al .
  • Cytosine-cleavage assays are based on the hydrolysis of phosphodiester bonds between cytosine nucleotides using either restriction enzymes or chemicals such as hydrazine (hydrazinolysis) or potassium permanganate (pyridinolysis) (Bickle and Kruger, 1993; Pfeifer et al . , 1989; Fritzsche et al., 1987).
  • restriction enzymes or chemicals such as hydrazine (hydrazinolysis) or potassium permanganate (pyridinolysis) (Bickle and Kruger, 1993; Pfeifer et al . , 1989; Fritzsche et al., 1987).
  • restriction enzymes with methylation-sensitive endonuclease activity at specific recognition sites are routinely used and applied to methods that incorporate either Southern hybridization and/or PCR detection.
  • Examples include methylation-restriction PCR (Singer-Sam et al., 1990), restriction landmark genomic scanning (RLGS) (Kawai et al . , 1994), differential methylation hybridization (DMH) (Huang et al . , 1999), methylation- sensitive arbitrarily primed PCR (AP-PCR) (Gonzalgo et al., 1997) and methylated CpG island amplification (MCA) (Toyota et al . , 1999). Most of these methods are useful where genomic or region-specific information is required when sequence data is not available.
  • Cytosine-conversion assays are based on the alkaline hydrolysis of double stranded DNA followed by the conditionally dependent deamination of sulphonated cytosines and 5-methylcytosines to uracil and thymine, respectively, using sodium bisulfite (Frommer et al . , 1992) .
  • rate of deamination are monitored to proceed only at sulphonated cytosines and not at 5-methylcytosines
  • unmethylated converted cytosines are distinguished as thymine by Taq polymerase from 5- methylcytosines as these remain unconverted.
  • Ms-SNuPE methylation-sensitive single nucleotide primer extension
  • MS-SSCA methylation-sensitive single-strand conformation analysis
  • MSP methylation-specific PCR
  • real-time MSP methods based on the PCR amplification of bisulfite modified DNA using probe- or dye-based fluorescence assays combined with LED strobes and photomultipliers offer significantly improved sensitivity and specificity over restriction enzymes , Southern hybridizations and conventional MSP (Higuchi et al . , 1992; Cottrell and Laird, 2003) .
  • Real-time kinetic analyses eliminate the inaccuracies of conventional endpoint amplicon quantification by detecting an initial inflection point in fluorescence signal which indicates the beginning of a log-linear phase in amplification on the exponential growth curve of PCR product.
  • a cycle threshold (C ⁇ ) value is determined and defined as the fractional cycle number at which a significant increase in fluorescence signal is detected above a fixed baseline threshold in fluorescence .
  • the C ⁇ is inversely proportional to the log initial starting copy number of DNA molecules present in the sample (Higuchi et al . , 1992) .
  • various alternative modes of detection, quantification and data normalization have been applied to real-time MSP based methods. Examples include various combinations of either hydrolysis or hybridization probes or fluorescent dyes as modes of detection (e.g.
  • the present invention provides a highly sensitive and accurate method for the absolute quantification of CpG methylation at any given genetic locus and further provides for the development of gene-specific diagnostic reagents. More specifically, the invention describes a novel systematic method to quantify the fractional ratio of preferentially targeted unmethylated to methylated CpG molecules or alleles at any given genetic locus .
  • the invention therefore allows the development of gene- specific diagnostic or prognostic tests based on the identification of CpG sites within the promoter region of a gene of interest that correlate with expression of the gene thus enabling identification of normal cell expression parameters and detection of disease related aberrations in gene expression.
  • the method provides for the absolute quantification of CpG methylation at the CBFA2T3 locus and further provides CBFA2T3-specific diagnostic reagents. More specifically, the invention describes a novel systematic approach to quantify the fractional ratio of preferentially targeted unmethylated to methylated molecules or alleles at the CBFA2T3 locus . The invention therefore allows the development of CBFA2T3-specific diagnostic or prognostic tests based on the identification of CpG sites within the promoter region of the gene that correlate with expression of the gene thus enabling identification of normal cell expression parameters and detection of disease related aberrations in CBFA2T3 gene expression.
  • the method when applied to a number of samples is preferably used to resolve : (1) a "methylation map" which defines the characteristic methylation pattern pertaining to a specific CpG island or sequence common to all samples on the basis of methylation memory; (2) a range of "normal cell methylation ratios” (defines the ratios of unmethylated versus methylated molecules [i.e.
  • the present invention provides a method for the identification of a normal cell methylation range, in order to allow for the detection of aberrant methylation, in a locus of interest in a nucleic acid, particularly at a CpG island, comprising the following steps : (a) providing separately the nucleic acids from a plurality of nucleic acid-containing samples and modifying said nucleic acids by cytosine-conversion; (b) identifying samples with high to moderately methylated CpG sites at the locus of interest; (c) generating a methylation map, particularly a CpG island map, from those samples identified in (b) above by sequencing cytosine-converted nucleic acids; (d) performing a targeted amplification of specific CpG sites as determined by the methylation map in (c) above from all samples by using nested real-time MSP and quantifying unmethylated to methylated ratios at these sites via extrapolation from absolute standard curves; and (e) establishing a correlation between
  • the present invention provides a method for the detection of aberrant methylation in a locus of interest in a nucleic acid, particularly at a CpG island, of a subject comprising the following steps: (a) providing the nucleic acids from a nucleic acid- containing sample from the subject for cytosine-conversion modification using sodium bisulfite; (b) performing a targeted ampli ication of at least one site at which abnormal methylation may occur by using nested real-time MSP and quantifying unmethylated to methylated ratios at these sites via extrapolation from absolute standard curves ; and (c) ascertaining whether the sample displays aberrant methylation relative to a normal cell methylation range established by the method described above.
  • the present invention provides a method for the diagnosis or prognosis of a disease in a subject, comprising the following steps: (a) providing the nucleic acids from a nucleic acid- containing sample from the subject and modifying said nucleic acids by cytosine-conversion ; (b) performing a targeted amplification of at least one site at which abnormal methylation may occur by using nested real-time MSP and quantifying unmethylated to methylated ratios at these sites via extrapolation from absolute standard curves ; and (c) ascertaining whether the sample displays aberrant methylation relative to a normal cell methylation range, as established by the method described above, in order to make a diagnosis and/or prognosis of the disease.
  • the method of this invention provides significant advantages over current methods used for assaying CpG methylation.
  • the method allows for the accurate absolute quantification of unmethylated and methylated CpG molecules or alleles at specific sites within any CpG island or gene under investigation. This in turn can allow for the identification of a normal population methylation range and thus the identification of abnormal methylation ranges associated with diseased samples . This approach as such provides the best way to determine the most accurate normal cellular methylation ratios and indices' .
  • the method provides for the identification of high frequency methylation sites for an accurate correlation with mRNA expression of the corresponding gene loci .
  • the method enables a reduction of PCR amplification bias as the method adopts the construction of standard curves that are representative of the efficiency of the oligonucleotide reagents used for sample testing.
  • Step (a) In this step, procedures for high quality nucleic acid isolation are used in order to maximize the complete removal of protein as this is known to interfere with the performance of subsequent cytosine-conversion treatments , typically using sodium bisulfite (Warnecke et al . , 2002).
  • intact genomic DNA is isolated using standard proteinase K/phenol extraction, guanidinium or anion exchange resin protocols .
  • the isolated DNA is then digested with several restriction enzymes such that all genomic DNA is digested except for the region of interest.
  • the DNA is then further purified, preferably by using silica-based spin columns .
  • Nucleic acid containing samples include, but are not limited to, bodily solids and fluids, normal tissue, diseased tissues (Wong et al . , 2000; Usadel et al . , 2002), blood or sputum (Belinsky et al . , 2002; Palmisano et al . ,
  • samples used for the analysis of the CBFA2T3 gene are derived from individuals with cancer.
  • these samples are obtained from individuals with breast cancer, prostate cancer, ovarian cancer, hepatocellular cancer, or primitive neuroectodermal cancer.
  • the cytosine-conversion assay is preferably conducted using sodium bisulfite solution whereby the revised optimal conditions for conversion are employed (Warnecke et al . , 2002) .
  • Strand separation is preferably accomplished and maintained under alkaline hydrolysis as routinely described, although, other means may been employed for strand separation such as heat denaturation (Rein et al . , 1997), the use of helicases and enzymes alike (Houston and Kodadek, 1994) , the addition of urea (Paulin, et al . , 1998), or by embedding the DNA in low melting agarose blocks (Olek et al. , 1996) .
  • Step (b) Following cytosine-conversion, the methylation status of a CpG island or sequence spanning at least 1 kb preferably within the promoter region or regulatory rich element of a gene is investigated, typically using MSP and/or restriction enzyme type analyses .
  • the CpG island may extend into the coding region of the gene such that this region also is analyzed.
  • CpG island prediction algorithms are often used in order to define the CpG island or sequence ( e . g. CpGfinder) .
  • the CpG island of the CBFA2T3 gene is investigated. Sequence incorporating this CpG island is represented by SEQ ID NO: 27 and is shown in Figure 9.
  • the CpG island under investigation is concomitantly examined for potential transcriptional activity by using assays such as CAT ELIZA.
  • assays such as CAT ELIZA.
  • the identification of samples harboring potentially methylated CpG sites within the island or DNA sequence of interest are initially screened for by using conventional MSP (Herman et al . , 1996).
  • the methylation status may be screened by standard restriction enzyme digestion and Southern hybridizations, MS-SSCA (Dobrovic et al., 2002), Ms-SnuPE (Gonzalgo and Jones, 1997) or COBRA (Xiong and Laird, 1997) .
  • oligonucleotide primers are designed to enable discrimination between unmethylated and methylated cytosines at the CpG position of interest.
  • oligonucleotide primers are designed such that they target at least 1-3 CpGs concentrated at the 3' end of the primer in order to minimize false positives due to mispriming events.
  • the primers should be designed such that they target enough orphan cytosines from the original sequence in order to minimize potential amplification of unmodified template.
  • oligonucleotide primer pairs used for analysis of the CBFA2T3 gene are lF/IRm, lF/lRu, 2F/2Rm, 2F/2Ru, 3F/3Rm, 3F/3Ru, 4F/4Rm and 4F/4Ru, sequences of which are provided in Table 1 and represented by SEQ ID NOs : 1-16 respectively.
  • the amount of DNA treated and subsequently analyzed is a parameter in terms of actually detecting potential methylation in a given specimen at specific CpG sites as shown by the following theoretical calculations : If we treat 1.0 ⁇ g (1 x 10 ⁇ pg) of genomic DNA with sodium bisulfite and assume that approximately 84-96% is degraded following conversion (Warnecke et al . , 2001), then this equates that approximately 40-160 ng of modified DNA is retrievable for analysis. This final concentration, which is also further based on an unlikely recovery rate of 100%, equates to approximately 1-3 reactions that can be performed by MSP if 40-50 ng is used per reaction as described routinely by most investigators .
  • Step (c) those samples displaying high to moderate methylation as identified by conventional MSP or equivalent methods are then used to generate a methylation map of the CpG island, typically by bisulfite primer amplification and sequencing.
  • bisulfite primers are designed such that they avoid regions containing CpG sites to enable the simultaneous amplification of both unmethylated and methylated molecules by using standard hot start PCR conditions and adequate concentrations of starting template as described.
  • Certain primers may require Y (C or T) or R (G or A) degeneracy in CpG rich regions .
  • the primers used should be designed to prevent false positives and generate amplicons preferably 100-300 bp in length to be subcloned and screened for positive clone inserts by colony PCR. On average, approximately 5- 10 positive clones are sequenced using conventional dideoxy termination sequencing methods . Several sequentially overlapping amplicons spanning the entire CpG island should be sequenced in order to monitor for potential bisulfite PCR amplification and cloning bias. Ideally, clones are sequenced as direct product sequencing can generate ambiguous chromatograms .
  • oligonucleotide primer pairs used for bisulfite sequencing of the CBFA2T3 gene are 1BF/1BR, 2BF/2BR, 3BF/3BR, and 4BF/4BR, sequences of which are shown in Table 1 and represented by SEQ ID NOs : 17-24 respectively.
  • Table 1 sequences of which are shown in Table 1 and represented by SEQ ID NOs : 17-24 respectively.
  • additional measures should be employed to prevent and monitor for false positives and amplification bias (Grunau et al . , 2001; Warnecke et al . , 2002).
  • the bisulfite modified DNA may be treated with restriction enzymes such as AIVLT prior to bisulfite amplification to shear potentially non-converted DNA (Steward et al .
  • the in s l ⁇ co template that represents the fully methylated and fully unmethylated sequence of the promoter region of the CBFA2T3 gene is provided as shown in Figure 10 (SEQ ID NO: 28) and Figure 11 (SEQ ID NO: 29) respectively.
  • the methylation map of the promoter region of the CBFA2T3 gene is provided as shown in Figure 3B .
  • Step (d) In this step, following generation of a methylation map, those amplicons testing negative for bisulfite PCR amplification and cloning bias are further amplified by second round real-time MSP.
  • the amplicon chosen harbors distinct CpG sites of frequent methylation within consensus recognition elements .
  • the designated amplicon is initially amplified by first round bisulfite PCR amplification from all samples and subsequently purified preferably using non gel-based silica purification spin columns .
  • the chosen amplicon for first round bisulfite PCR amplification of the CBFA2T3 gene promoter is generated using the 2BF/2BR oligonucleotide primer pair, sequences of which are shown in Table 1 and represented by SEQ ID NOs : 19 and 20 respectively .
  • the purified amplicons are then re-amplified by realtime PCR using SYBR Green I detection to enable melt curve comparisons and concentration equilibration.
  • These amplicons are subsequently amplified by second round realtime MSP using internal MSP and USP primers that target frequently methylated regulatory CpG sites (as identified by bisulfite sequencing) and unmethylated CpG sites respectively.
  • the oligonucleotide primers are designed such that they target at least 1-3 CpGs concentrated at the 3' end and are used in combination with an original bisulfite primer or secondary internal nested bisulfite primer.
  • the oligonucleotide primer pairs chosen for second round realtime MSP of the CBFA2T3 gene promoter are mF/2BR (SEQ ID NOs: 25 and 20 respectively) and uF/2BR (SEQ ID NOs: 26 and 20 respectively) , sequences of which are shown in Table 1.
  • two internal bisulfite primers and a secondary internal primer or probe e . g.
  • Scorpion primer TaqMan, Molecular Beacon or dual-labeled FRET
  • an internal Scorpion MSP/USP primer combination and an internal bisulfite primer combination would be used (Thelwell et al . , 2000) .
  • primers designed and validated to bind only non-converted or partially converted DNA may be multiplexed in order to remove background non-specific amplification.
  • any possible combinations in use should be optimized prior to specimen analysis by for example performing amplifications in real-time on dilutions of consensus unmethylated and methylated clones using methylated and unmethylated-specific primer or probes respectively.
  • unknown MSP and USP molecule numbers are extrapolated from standards of purified amplicons of consensus methylated or consensus unmethylated clones, respectively.
  • dsDNA methylated and unmethylated amplicons are 10-fold serially diluted from 10 ng/ ⁇ l to 10 ag/ ⁇ l .
  • the total M.W. of each standard amplicon is calculated from both sense and anti-sense strands .
  • Molecules/ ⁇ l of purified amplicons are then calculated from the mass in ⁇ g/ ⁇ l of amplicons ⁇ MW (x 6.02 x 10 17 mlcls/ ⁇ mole) .
  • a 100% efficient amplification means a doubling of amplification product in each cycle resulting in a slope of -3.322, an amplification factor of 2 and a reaction efficiency of 1.
  • a slope of 3.8 means the reaction has an amplification value of ⁇ 1.83 and a reaction efficiency of 0.83 or 83%.
  • a relative means for quantification such as comparative cycle threshold ( ⁇ C T ) calculations can be used to normalize for di ferential reaction ef iciencies .
  • This approach is useful to support absolute data, however, as the comparative C ⁇ method compares the target amplicon to an internal reference amplicon (normalizer) in order to calculate relative copy numbers, this method is time- consuming. It requires the dynamic range in amplification efficiencies of target and reference amplicons to be similar enough over a dilution series to enable valid quantification (PE Applied Biosystems, User bulletin no. 2: 1-36, 1997). Nonetheless, any extrapolation curves used are preferably amplified prior to sample analysis and are saved as template files for routine extrapolation.
  • Step (e) Having accumulated independent real-time MSP and RT- PCR data sets from the samples under investigation it is then possible to plot these data and derive a regression equation that predicts the relationship between CpG methylation and corresponding mRNA expression.
  • This approach enables the independent variable analysis of alternative data sets which can be used to solve an unknown x or y. Where several samples are available this approach can the be used to identify coordinates which define a "normal cellular methylation-expression index" which may have widespread applicability for the gene of CpG sites under investigation.
  • the method of the invention also provides for the basis of a kit useful for the routine screening of samples to identify aberrant methylation and expression of any gene of interest for disease diagnosis and/or prognosis .
  • the kit may include components such as : (1) protocols recommended for obtaining DNA from a test sample for first round methylation specific PCR (MSP) of the gene of interest (such as DNA isolation from the sample, digestion of the DNA with restriction enzymes , and bisulfite modification of the DNA) ; (2) primers to amplify first round MSP products from the DNA obtained in (1) ; (3) primers to enable second round real-time MSP amplification from serial dilutions of cleaned first round MSP products; (4) serial dilutions of second round real-time MSP amplicons from a control sample to generate standard curves for extrapolation of methylation levels from the sample being tested; (5) a regression equation for the generation of standard curves to predict the relationship between aberrant promoter methylation of the gene of interest and mRNA expression of the gene in the test sample; and (6) a normal cellular methylation level defined by a range of methylation indices as predetermined for the gene of interest and provided in the kit.
  • MSP methylation specific
  • the kit may also include the methylation map for the gene of interest so that the choice of primers for testing the methylation status of the gene can be determined by the investigator.
  • the method of this invention has been put into practical utility through the analysis of the promoter region of isoform b of CBFA2T3.
  • a kit comprising reagents and resources for the routine screening of samples for CBFA2T3 aberrant methylation and expression for disease diagnosis and/or prognosis .
  • the kit may include components such as : (1) protocols recommended for obtaining DNA from a test sample for first round methylation specific PCR (MSP) of the CBFA2T3 gene (such as DNA isolation from the sample, digestion of the DNA with restriction enzymes, and bisulfite modification of the DNA) ; (2) primers to amplify first round MSP products from the DNA obtained in (1) .
  • MSP methylation specific PCR
  • primers 2BF (SEQ ID NO: 19) and 2BR (SEQ ID NO: 20) as shown in Table 1
  • primers to enable second round real-time MSP amplification from serial dilutions of cleaned first round MSP products .
  • primers mF (SEQ ID NO: 25) and 2BR (SEQ ID NO: 20) for amplification of methylated DNA for amplification of methylated DNA
  • primers uF (SEQ ID NO: 26) and 2BR (SEQ ID NO: 20) for amplification of unmethylated DNA for example primers mF (SEQ ID NO: 25) and 2BR (SEQ ID NO: 20) for amplification of methylated DNA
  • primers uF (SEQ ID NO: 26) and 2BR (SEQ ID NO: 20) for amplification of unmethylated DNA for example primers mF (SEQ ID NO: 25) and 2BR (SEQ ID NO: 20) for amplification of methylated DNA
  • primers uF (SEQ ID NO: 26) and 2BR (SEQ ID NO: 20) for amplification of unmethylated DNA for example primers mF (SEQ ID NO: 25) and 2BR (SEQ ID NO: 20) for amplification of methylated DNA, and
  • the kit may also include the methylation map for the CBFA2T3 gene such as that shown in Figure 3B so that the choice of primers for testing the methylation status of the gene can be determined by the investigator .
  • an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 27. This sequence corresponds to the wild-type promoter sequence of the CBFA2T3 gene as shown in Figure 9.
  • an isolated nucleic acid molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 27.
  • nucleic acid sequence of the fully methylated CBFA2T3 promoter as represented by SEQ ID NO: 28 and shown in Figure 10
  • nucleic acid sequence of the fully unmethylated CBFA2T3 promoter as represented by SEQ ID NO: 29 and shown in Figure 11.
  • most genes here displayed up- or down-regulation within 15-fold in breast tumor cell lines (Bieche et al . , 1999; Beijersbergen et al . , 1994) . FVI values are shown in brackets .
  • SYK displayed highly aberrant expression as shown in (C) . Greater than 300-fold down-regulation was detected in BT-5 9, MDA-MB-231 and MDA-MB-157. Greater than 50-fold up-regulation was detected in DU4475.
  • DU4475, T47D, MDA-MB-134 and MDA-MB-453 has been shown to correlate with methylation (Hui et al . , 2000; Bisogna et al., 2001). Greater than 10-fold up-regulation in MB436, MDA-MB-157, MDA-MB-415, HBL100, MDA-MB-468 and BT-549 is consistent with previous data (Musgrove et al . , 1995; Hui et al., 2000; Bisogna et al . , 2001). P53 displayed relatively uniform expression compared to SYK and INK4A/ARF as shown in (C) . Expression ranged 40-fold (5 to 200 mRNA mlcls/cell) .
  • Altered expression of P53 in breast tumors may result from differential promoter usage or transcript variants (Thompson et al . , 1992; Raman et al. , 2000) .
  • BRCA1 displayed uniform expression in most cell lines except for HBL100 as shown in (C) . Expression ranged 600- fold from 5 x 10 "4 to 0.3 mRNA mlcls/cell in SKBR3 and HBL100 respectively.
  • Over-expression in HBL100 has been shown previously (Dimitrov et al . , 2001; Esteller et al . , 2000; Favy et al . , 2000).
  • Figure 3. CBFA2T3b promoter CpG island and bisulfite sequence analysis.
  • (A) 161 CpG sites spanning 1115 bp scaled as single bars relative to the transcription start exon lb. CAT constructs, MSP, bisulfite primers, CpG island and predicted promoter sites are shown.
  • (B) Periodic sinusoidal CpG hypermethylation scaled relative to the CBFA2T3h CpG island.
  • (C) 161 CpG sites spanning 1115 nucleotides were analyzed by bisulfite sequencing in selected breast tumor cell lines, primary breast tumors and normal bloods . Protected m 5 CpGs were scored as percent cytosines methylated/5-10 clones. Full methylation maps are shown for cell lines only.
  • Unmethylated (U) and methylated (M) band intensities were scored as high (black dot) , low (gray dot) , or negative (white dot) .
  • B) Low-level basal methylation defined by high unmethylated to low methylated band intensities was characteristic of CBFA2T3h promoter methylation throughout all regions in normal blood, cell lines and tumor/normals . The pUC19 DNA/ spI size markers equate to an approximate ratio of 100:1 unmethylated to methylated CBFA2T3b promoter mlcls .
  • C The general score for complex basal and high methylation is shown for primary breast tumor/normals region four.
  • CBFA2T3b promoter melt peak analyses Melt peaks of mF/2BR and UF/2BR amplicons in normal bloods and breast tumor cell lines . Normal bloods displayed consistent -dF/dT m levels in both unmethylated and methylated molecules compared to aberrant -dF/dT m levels in breast tumor cell lines .
  • Figure 7. CBFA2T3b promoter methylation versus CBFA2T3 exon 4-5 mRNA expression.
  • (B) CBFA2T3b promoter methylation versus mRNA expression calculated per 10 4 cells for breast tumor cell lines with known 16q24.3 DNA mlcls/cell.
  • Greater than 1000-fold up-regulation was also detected in spleen tyrosine kinase (SYK) expression, a tumor suppressor previously shown to be hypermethylated and down-regulated in MDA-MB-231 cells (Yuan, et al . , 2001) .
  • Figure 9. Provides the wild-type nucleic acid sequence of the region surrounding the CBFA2T3 isoform b promoter .
  • a total of approximately 5kb incorporating exon lb is shown indicating the high density distribution of CpG dinucleotides .
  • CpG dinucleotides are highlighted in bold. The sequence is in a 5' to 3' orientation.
  • Figure 10. Provides the sequence of the fully methylated in silico prediction of the. CBFA2T3 isoform b promoter region following complete cytosine conversion using sodium bisulfite.
  • the protected CpG dinucleotides are shown in bold.
  • the sequence is in a 5' to 3' orientation.
  • Figure 11 Provides the sequence of the fully unmethylated in silico prediction of the CBFA2T3 isoform b promoter region following complete cytosine conversion using sodium bisulfite.
  • the unprotected CpG dinucleotides are shown in bold. The sequence is in a 5' to 3' orientation .
  • Example 1 Absolute quantification of mRNA expression by real-time RT-PCR Cytogenetic aberrations reported at 16q24.3 in primary breast tumors, preinvasive DCIS and ductal hyperplasia, denotes the location of key regulatory gene(s) or loci involved in the initiation and development of sporadic breast tumorigenesis (Cleton-Jansen et al . , 2001). From a recent 16q24.3 mRNA expression survey CBFA2T3 was revealed to display highly aberrant expression in breast tumor cell lines and therefore confers a likely candidate (Powell et al . , 2002). These initial studies demonstrated aberrant expression of CBFA2T3 exon 4-5 mRNA in breast tumor cell lines.
  • CYPA cyclophilin A
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • RNA Contamination was visualized by SYBR Green I (BMA) staining on agarose gels . Ribosomal RNA integrity was confirmed using SYBR Green I . Total RNA concentrations were determined by A 2 ⁇ o readings and RiboGreen quantitation (Molecular Probes) .
  • RNA (10 ⁇ g at > 1.0 ⁇ g/ ⁇ l) was oligo (dT) 16 reverse transcribed at 55°C for 2 h using MMLV (Promega) with addition of RNAguard (Promega) and DMSO.
  • cDNA:RNA hybrids were hydrolyzed at 37°C for 30 min using RNase H (Promega).
  • CYPA-1 , CBFA2T3 exon la and exon lb amplicons were quantified using TaqMan probes (Geneworks) .
  • C ⁇ PA-2 , CBFA2T3 exon 4-5, ATP5A and SYK were quantified using SYBR Green I (BMA) .
  • Primers and probes were designed using PrimerSelect (DNAStar) and BeaconDesign (PremierBiosoft) . Probes were analyzed independently to avoid predicted secondary structure. Reactions were performed on a Rotor-Gene 2000 (Corbett Research) . Final reactions contained IX buffer, 200 nM each dNTP, 1.5-4.0 mM MgCl 2 , 200-500 nM each primer, 0.25U HotStar Taq (Qiagen) , 0.35X SYBR Green I or 100-200 nM probe and cDNA equivalent to 100-1500 ng total RNA.
  • Amplifications were at 95°C for 10 min, 45 cycles at 94°C for 20 s, T A (see Table 1) for 30 s and 72°C for 30 s. Product specificity was confirmed by melt curve analysis and SYBR Green I. Fluorescence data was acquired at 72°C for SYBR and 60°C for TaqMan probes .
  • cDNA amplicons were generated from normal tissue cDNA using HotStar Taq and purified using QIAquick (Qiagen) . Concentrations (routinely > 50 ng/ ⁇ il) were determined by A 26 o readings and RiboGreen quantitation. Molecules (mlcls) / ⁇ l of purified amplicons were calculated from ⁇ g/ ⁇ l of amplicons ⁇ MW (x 6.02 x 10 17 mlcls/ ⁇ mole) .
  • Amplicons were 10-fold serially diluted from 10 ng/ ⁇ l to 10 ag/ ⁇ l .
  • Unknown mlcl numbers were extrapolated at the C ⁇ (cycle threshold) from standard curves with known mlcls/ ⁇ l. Standard curves were amplified during or prior to sample analysis .
  • Unknown molecule numbers were extrapolated at the C ⁇ (cycle threshold) from standard curves with known mlcls/ ⁇ l.
  • mRNA mlcls/pg of total RNA were calculated from C ⁇ concentration ⁇ amount of total RNA (pg) per reaction.
  • mRNA mlcls/cell were calculated from mRNA mlcls/pg x 4 based on a 4 pg total RNA/cell estimation .
  • Expression levels were extrapolated from replicate standard curve assays (n ⁇ 2) with C ⁇ coefficients of variation averaging ⁇ 15% over six orders within replicates and between dilutions .
  • Absolute expression levels were calculated from raw expression data (n > 4) and/or monitored against CYPA or ATPase coupling factor 6 subunit (ATP5A) .
  • CBFA2T3 mRNA expression The amplicon spanning exons 4-5 is common to both variants and was considered to estimate the total amount of CBFA2T3 mRNA.
  • CBFA2T3 exons la, lb and 4-5 displayed similar expression profiles in breast tumor cell lines ( Figure 1A) .
  • Exon la was expressed at 65-fold lower than the total exon 4-5.
  • exon lb was only 10- fold lower than exon 4-5, thus indicating that CBFA2T3b is the predominant variant.
  • the sum of la + lb expression averaged 9-fold lower than the expected total exon 4-5. 5'-UTR RNA degradation and/or assay variation may explain this .
  • CBFA2T3 exon 4-5 expression ranged 30, 000- old from 4 mRNA mlcls/10 4 cells in MDA-MB-231 to 120,000 mRNA mlcls/10 4 cells in BT-483. In contrast, uniform CYPA and ATP5A expression ranged 2-fold (100 to 200 mRNA mlcls/cell) and 20-fold (15 to 300 mRNA mlcls/cell) . CBFA2T3 exon 4-5 expression was examined in primary breast tumor for which total RNA was available . Relative to normal breast, CBFA2T3 was up-regulated by > 10-fold in 35% of primary tumors with no significant down- regulation detected (Figure IC) .
  • GenBank accession numbers Cyclophilin A (CYPA; 30167) , glyceraldehyde-3- phosphate dehydrogenase ( GAPDH; M33197) , core binding factor, alpha subunit 2; translocation to, 3 (CBFA2T3;
  • TRADD TRADD; NM_003789
  • E2F transcription factor 4 E2F4; NM_001950
  • MTGR1 yeloid translocation gene 8-like protein
  • PIAS2 protein inhibitor of activated STAT1
  • MYC c-myc oncogene
  • Example 2 Identification of basal methylation by methylation-specific PCR (MSP) Since CBFA2T3b was found to be the predominant variant displaying aberrant expression in breast tumor cell lines, we reasoned the promoter driving expression of this variant may become deregulated in breast tumors via aberrant CpG methylation, a mechanism now described for several tumor suppressors and oncogenes (Jones and Baylin, 2002) . The transcription potential of a predicted promoter and CpG island 5' to CBFA2T3 exon lb was assessed by CAT analysis.
  • MSP methylation-specific PCR
  • the 1087 bp B sequence contained cis-acting elements capable of inducing a 30-fold increase in CAT expression ( Figure 3D) .
  • Figure 3D To determine if aberrant CpG methylation of the CBFA2T3b promoter occurs in breast tumors, four separate 100-200 bp regions spanning 1 kb of promoter sequence were examined by methylation-specific PCR (MSP) .
  • MSP methylation-specific PCR
  • HMECs were from Clonetics . Genomic DNA was isolated using GenElute for mammalian tissue (Sigma) or QIAamp for blood (Qiagen) .
  • DNA was desalted using Qiagen purification columns, eluted in 50 ⁇ l H 2 0 and desulfonated with 5.5 ⁇ l of 3 M NaOH for 20 min at 37°C. DNA was neutralized and precipitated with 80 ⁇ l of 10 M ammonium acetate (pH 7.0) , 2 ⁇ l linear acrylamide and 500 ⁇ l cold 100% EtOH . Modified DNA was resuspended in 20 ⁇ l 1 mM Tris-Cl (pH 8.0) and stored at -80°C.
  • CBFA2T3b promoter basal methylation Four separate 100-200 bp regions spanning 1 kb of promoter sequence were examined by methylation-specific PCR (MSP) on > 300 ng of bisulfite modified DNA using standard HotStar Taq reaction conditions (Qiagen) . Optimal primers (PrimerSelect) , annealing temperatures and sequence locations are shown (Table 1, Figure 3A) . Unmethylated and methylated band intensities were scored as high, low or negative in normal bloods, breast tumor cell lines and primary breast tumor/normals ( Figure 4A) . Low-level basal methylation defined by high unmethylated to low methylated band intensities was detected in all normal bloods in > 2/4 regions.
  • MSP methylation-specific PCR
  • Example 3 Generation of a methylation map by bisulfite sequencing
  • specific m 5 CpG sites within the CBFA2T3b promoter could be targeted by real-time MSP to accurately distinguish hypo- and hyper-methylation from basal methylation and clearly resolve a correlation between aberrant CpG methylation and mRNA expression .
  • a methylation map spanning 161 CpG sites within 1115 bp of CBFA2T3b promoter sequence was generated by bisulfite sequencing .
  • Bisulfite genomic sequencing Several bisulfite primers were designed to amplify both unmethylated and methylated mlcls for methylation pattern analysis. Optimal primers (Pri erSelect) , annealing_ temparatures and_ sequence locations are shown (Table 1, Figure 3A) . Amplicons were generated from > 300 ng of bisulfite modified DNA using optimized HotStar Taq reaction conditions (Qiagen) . Amplicons were cloned into pGEM (Promega) and an average 5-10 clones/amplicon sequenced using BigDye (PE Biosystems) . Note here that all MSP and bisulfite primers were tested on unmodified and modified DNA to confirm for amplification specificity of cytosine conversion.
  • CBFA2T3b promoter methylation map Percent methylation frequencies/5-10 clones for selected cell lines, tumor/normals and normal bloods are shown in Figure 3C .
  • Example 4 Absolute quantification of 5 CpG:m 5 CpG methylation ratios by real-time MSP Based on these methylation maps , primers spanning CpG sites -452, -444, -442 and -439 relative to exon lb were designed for real-time MSP. These cytosines were targeted because they located within an amplicon negative for bisulfite PCR bias, span Spl and homeotic recognition elements , re-amplified specifically by second round MSP and were hypermethylated in breast tumors .
  • Real-time MSP 2BF/2BR (SEQ ID NOs: 19 and 20 respectively) amplicons for all samples were generated from > 300 ng of bisulfite modified DNA using optimized HotStar Tag- reaction conditions (Qiagen) and column purified (Qiagen) . Methylated to unmethylated ratios at CpGs -452, -444, -442 and -439 relative to exon lb were quantified by second round real-time SYBR-MSP on 2BF/2BR amplicons using mF/2BR (SEQ ID NOs: 25 and 20 respectively) and uF/2BR (SEQ ID NOs: 25 and 20 respectively) and uF/2BR (SEQ ID
  • UF/2BR molecule numbers were extrapolated from standards of purified amplicons generated from fully methylated (MDA-MB-231) and fully unmethylated (normal blood or BT- 483) clones respectively.
  • dsDNA amplicons were 10-fold serially diluted from 10 ng/ ⁇ l to 10 ag/ ⁇ l .
  • Unknown mlcl numbers were extrapolated at the parameter C ⁇ (cycle threshold) from standard curves with known mlcls/ ⁇ l. Standard curve reaction efficiencies (methylated- 96%, unmethylated- 72%) corrected for quantification of absolute unmethylated to methylated ratios .
  • the first-round 2BF/2BR amplicons were re-amplified by real-time SYBR detection for melt curve analyses and concentration equilibration to permit quantification and extrapolation of unknown sample mF/2BR and UF/2BR concentrations within the standard curve linear range .
  • mF/2BR and UF/2BR primer specificities were tested by real-time amplification on dilutions of unmethylated and methylated clones respectively.
  • mF/2BR and UF/2BR amplicons generated from real-time MSP were sequenced to ensure primer specificity.
  • CBFA2T3b promoter methylation ratios Absolute ratios of unmethylated to methylated CpGs - 452, -444, -442 and -439 were quantified by second round real-time MSP on 2BF/2BR amplicons using internal mF and uF primers . Unmethylated to methylated CpG ratios (u/m) for normal bloods, breast tumor cell lines and primary breast tumor/normals were quantified at the parameter C ⁇
  • breast tumor cell lines 75% were aberrantly methylated outside the range of normal blood basal methylation. 58% were outside the range of normal breast. Half of these aberrations were either hyper- or hypo-methylated relative to both normal blood and normal breast. Similar to cell lines, 51% of primary breast tumors were aberrantly methylated relative to normal blood. 35% were aberrant relative to normal breast, i . e . 24% were hypermethylated and 11% hypomethylated. Aberrant methylation ratios in breast tumors ranged from 1:10 unmethylated to methylated molecules in hypermethylated MDA-MB-231 to 7000:1 in hypomethylated BT-483.
  • CBFA2T3b promoter melt peak analyses Melt peaks of mF/2BR and uF/2BR amplicons in normal bloods and breast tumor cell lines were generated following real-time MSP amplification ( Figure 6) . Peaks were calculated from the negative derivative in fluorescence over temperature verses temperature (-dF/dT m versus !T m ) . Normal bloods displayed consistent -dF/dT m levels in both unmethylated and methylated molecules . In contrast, breast tumor cell lines displayed aberrant dF/dT m levels depicted by broad melt transitions and heterogeneous melt peaks caused by an aberrant concentration rather than composition of m 5 CpGs .
  • Example 5 Correlation between promoter methylation and mRNA expression
  • a regression equation was derived that predicts the relationship between aberrant CBFA2T3b promoter methylation and mRNA expression.
  • Methylation indices were further expressed as methylated CBFA2T3b promoter mlcls/10 4 cells for breast tumor cell lines and plotted against total CBFA2T3 mRNA mlcls/10 4 cells (Figure 7B) .
  • Example 6 Activation of CBFA2T3b mRNA expression by 5- Aza-dC and TSA An association between promoter hypermethylation and reduced CBFA2T3b mRNA expression in MDA-MB-231 was further established using demethylating agents 5-Aza-dC and TSA.
  • 5-Aza-dC and TSA assay Breast tumor cell line MDA-MB-231 was seeded at 2.0 x 10 5 cells/T75 flask in 10 ml RPMI-1640 supplemented with 10% fetal calf serum, 15 mM HEPES, 10 mg/liter penicillin/streptomycin and cultured for 48 h at 37°C with 5% C0 2 .
  • CBFA2T3b isoform can be alleviated using demethylating agents and compensated via retroviral re-expression to mediate growth suppression (Kochetkova et al . , 2002).
  • T A Annealing temperature of primers and probe.
  • C to U to T conversions mSlicated as bold type.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé d'identification d'un intervalle de méthylation de cellules normales, qui permet de détecter la méthylation aberrante, dans un site cible d'un acide nucléique, en particulier dans un îlot CpG. Ce procédé consiste à: (a) fournir séparément les acides nucléiques provenant d'une pluralité d'échantillons renfermant des acides nucléiques et modifier ces acides nucléiques par conversion de cytosine; (b) identifier les échantillons qui possèdent des sites CpG présentant une méthylation élevée à modérée dans le locus cible; (c) produire une carte de méthylation, en particulier une carte de l'îlot CpG, à partir des échantillons précédemment identifiés en (b) par séquençage des acides nucléiques ayant subi une conversion par cytosine; (d) exécuter une amplification ciblée des sites CpG spécifiques tel que déterminé par la carte de méthylation en (c), à partir de tous les échantillons, par MSP imbriqué en temps réel, et quantifier les rapports de non méthylation et de méthylation dans ces sites via l'extrapolation de courbes d'étalonnage absolu; et enfin, (e) établir une corrélation entre la méthylation dans ces sites CpG spécifiques et les taux d'expression d'ARNm correspondant tel que déterminé par une méthode RT-PCR en temps réel.
PCT/AU2004/001398 2003-10-14 2004-10-14 Procede de diagnostic base sur la methylation WO2005038046A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2003905593A AU2003905593A0 (en) 2003-10-14 Methylation-based diagnostic method
AU2003905593 2003-10-14

Publications (1)

Publication Number Publication Date
WO2005038046A1 true WO2005038046A1 (fr) 2005-04-28

Family

ID=34437865

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2004/001398 WO2005038046A1 (fr) 2003-10-14 2004-10-14 Procede de diagnostic base sur la methylation

Country Status (1)

Country Link
WO (1) WO2005038046A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002070742A1 (fr) * 2001-03-01 2002-09-12 Epigenomics Ag Procede de mise au point de groupes d'echantillons de genes a des fins de diagnostic et de therapie qui sont bases sur l'expression et l'etat de methylation des genes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002070742A1 (fr) * 2001-03-01 2002-09-12 Epigenomics Ag Procede de mise au point de groupes d'echantillons de genes a des fins de diagnostic et de therapie qui sont bases sur l'expression et l'etat de methylation des genes

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ASADA K. ET AL.: "Reduced expression of GNA11 and silencing of MCT1 in human breast cancers", ONCOLOGY, vol. 64, May 2003 (2003-05-01), pages 380 - 388 *
BAIS A.J. ET AL.: "Aberrant CBFA2T3B gene promoter methylation in breast tumours", MOLECULAR CANCER, vol. 3, no. 22, August 2004 (2004-08-01) *
KANEDA A. ET AL.: "Reduced expression of the insulin-induced protein 1 and P41 ARP2/3 complex genes in human gastric cancers", INTERNATIONAL JOURNAL OF CANCER, vol. 1000, 2002, pages 57 - 62 *
MIYAMOTO K. ET AL.: "Methylation-associated silencing of herpan sulfate D-glucosaminyl 3-O-sulfotransferase-2(3-OST-2) in human breast, colon, lung and pancreatic cancers", ONCOGENE, vol. 22, January 2003 (2003-01-01), pages 274 - 280 *
PALMISANO W.A. ET AL.: "Aberrant promoter methylation of the transcription factor genes PAX5 alpha and beta in human cancers", CANCER RESEARCH, vol. 63, August 2003 (2003-08-01), pages 4620 - 4625 *
TAKAI D. ET AL.: "Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis", ONCOGENE, vol. 20, 2001, pages 7505 - 7513 *
TOYOTA M. ET AL.: "Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification", CANCER RESEARCH, vol. 59, 1999, pages 2307 - 2312 *

Similar Documents

Publication Publication Date Title
Derks et al. Methylation‐specific PCR unraveled
Paz et al. Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases
Zardo et al. Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors
US8088581B2 (en) Differential enzymatic fragmentation by whole genome amplification
Rauch et al. MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells
Shi et al. Oligonucleotide‐based microarray for DNA methylation analysis: principles and applications
Weber et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells
Shi et al. Expressed CpG island sequence tag microarray for dual screening of DNA hypermethylation and gene silencing in cancer cells
Dahl et al. DNA methylation analysis techniques
Watanabe et al. Methylation of DNA in cancer
Toyota et al. Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification
AU2019201419B2 (en) A method of screening for colorectal cancer
US20070259368A1 (en) Gastric cancer biomarker discovery
EP1693468A1 (fr) Procédé de détection de l'état de méthylation d'un acide polynucléique
EP3594366B1 (fr) Panneau de marqueur de gènes de diagnostic
Costello et al. Aberrant methylation of genes in low-grade astrocytomas
US7932027B2 (en) Method for determining the methylation pattern of a polynucleic acid
US20090162836A1 (en) Prognostic and Diagnostic Markers for Cell Proliferative Disorders of The Breast Tissues
Mund et al. Array-based analysis of genomic DNA methylation patterns of the tumour suppressor gene p16 INK4A promoter in colon carcinoma cell lines
WO2002081749A2 (fr) Sequences d'adn a methylation modifiee utilisees comme marqueurs associes au cancer humain
Nishiyama et al. Both hypomethylation and hypermethylation in a 0.2-kb region of a DNA repeat in cancer
EP2410064A1 (fr) Procédés à haut rendement comprenant l'analyse d'une méthylation d'ADN à éléments répétitifs
WO2003035860A1 (fr) Methode d'identification de genes fondee sur une methylation differentielle de l'adn
Dahl et al. A ligation assay for multiplex analysis of CpG methylation using bisulfite-treated DNA
CN109385465B (zh) 一种dna甲基化定量系统

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase