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WO2007137597A1 - Tests destinés à la détection de mutations de points chauds et de la méthylation du gène 2 de type rétinoblastome (rbl2) utilisées comme marqueurs diagnostiques et pronostiques de tumeurs - Google Patents

Tests destinés à la détection de mutations de points chauds et de la méthylation du gène 2 de type rétinoblastome (rbl2) utilisées comme marqueurs diagnostiques et pronostiques de tumeurs Download PDF

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WO2007137597A1
WO2007137597A1 PCT/EP2006/005019 EP2006005019W WO2007137597A1 WO 2007137597 A1 WO2007137597 A1 WO 2007137597A1 EP 2006005019 W EP2006005019 W EP 2006005019W WO 2007137597 A1 WO2007137597 A1 WO 2007137597A1
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methylation
rbl2
gene
cpg
dna
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Caterina Cinti
Dario La Sala
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Consiglio Nazionale delle Richerche CNR
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    • 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

Definitions

  • the invention relates generally to cancer markers, and more specifically to cancer markers obtained from CpG rich regions of genes that are susceptible to methylation, in order to allow evaluating a gene methylation within a sample, and to detect conditions associated with the methylation status of a gene.
  • DNA methylation As known, DNA methylation in eukaryotic cells has profound effects on the genome. DNA methylation is an epigenetic modification of DNA itself through the transfer of a methyl group to the carbon 5 position of cytosine. This occurs usually in the context of CpG dinucleotides, i.e sites in the DNA where a cytosine nucleotide is next to a guanine nucleotide.
  • CpG islands are frequently located within the promoter regions of human genes and methylation within the islands has been shown to be associated with transcriptional inactivation of the corresponding gene
  • Some of the effects of methylation on the mammalian genome include transcriptional repression, chromatin structure modulation, X chromosome inactivation, genomic imprinting and the suppression of the detrimental effects of repetitive and parasitic DNA sequences on genome integrity (Baylin S. B. and Herman J. G., Trends Genet. 16, 2000) .
  • DNA methylation is clearly important during development; however, the requirement for DNA methylation in adult tissue might be much lower and its primary role might be to maintain the bulk, non-coding, portion of genome in a transcriptionally inactive state, effectively increasing the specificity of transcription factors for their target sites within genes. Methylation of CpG-rich islands in regulatory regions of genes marks them for transcriptional silencing. However, in normal tissue, for the majority of genes associated with CpG islands, the islands remain methylation-free regardless of whether the gene is expressed or not.
  • tumor suppressor genes can be inactivated by both genetic and epigenetic events thus favoring tumorigenesis .
  • Cancer is a stepwise process of accumulation of genetic and epigenetic abnormalities that can lead to cellular dysfunction; a synergy between these two processes drives tumor progression and malignancy. Genomic methylation patterns are frequently altered in tumor cells with global hypomethylation accompanying region-specific hypermethylation events. This can lead to genetic instability and to the repression of tumor suppressor genes.
  • methylated CpG islands reside within repetitive elements, inhibiting their activity.
  • the loss of methylation in tumor cells may predispose them to genomic instability, via the transcriptional activation and movement of parasitic elements and/or to genome rearrangement, via mitotic recombination, which could inactivate critical growth regulatory genes, or through whole chromosome arm gain and/or loss. All these mechanisms result in an inappropriate level of expression of a tumor suppressor gene or growth-promoting gene.
  • De novo methylation of 'CpG islands' in the promoter regions of tumor suppressor genes may lead to transcriptional silencing through a complex process involving histone deacetylation and chromatin condensation, and thus represents a tumorigenic event that is functionally equivalent to genetic changes, like mutation and deletion (Jones P. A. and Laird P. W., Nat. Genet. 21, 1999).
  • the genes affected include over half of the tumor suppressor genes that cause familial cancers when mutated in the germ line.
  • the selective advantage for genetic and epigenetic dysfunction in these genes is very similar.
  • the promoter methylation may be the only type of gene inactivation found in human cancer, since mutations for many of genes are rare or have not been observed.
  • the aberrant methylation can begin very early in tumor progression and mediate most of the important pathway abnormalities in cancer including loss of cell cycle control, altered function of transcription factors, altered receptor function, disruption of normal cell-cell and cell-substratum interaction, inactivation of signal transduction pathways, loss of apoptotic signals and genetic instability.
  • the profile of promoter hypermethylation for the genes differs for each cancer type, providing a tumor-type and gene-specific profile. This is consistent with a model in which methylation of CpG islands at particular genes would give to the cancer cell a growth or survival advantage and so aberrant patterns of methylation emerge depending on the selective pressure for gene silencing in the tumors type examined (Hanahan D. and Weinberg R. A., Cell 100, 2000).
  • Some genes such as the cell cycle inhibitor pl ⁇ INK4a, are hypermethylated across many tumor types, including colorectal, lung, and breast carcinomas (Sanchez-Cespedes M. et al., Cancer Res. 60, 2000; Esteller M. et al., Cancer Res. 60, 2000). Moreover, in any given tumor it is possible to find simultaneous inactivation of several pathways by aberrant methylation compromising either cell survival or tumor progression genes.
  • retinoblastoma-like 2 gene also called RBL2 or pl30
  • RBL2 or pl30 a negative cell cycle regulator
  • both global hypomethylation and regional hypermethylation are two independent processes that may coexist in a same cell. Both global hypomethylation and regional hypermethylation confer a selective advantage upon cancer cells by targeting different sets of genes with opposing roles in cellular transformation. Regional hypermethylation may target the silencing of genes that suppress tumorigenesis, whereas global hypomethylation may target activation of genes that are required for different stages of the transformation process.
  • a detection of methylated genes serves as biomarker for early detection of cancer, for risk assessment and for predicting response to therapy. Since methylation changes often appear early in disease, detection of hypermethylated genes could identify tissues derived from a subject with increased risk of cancer. Furthermore, the reversible nature of methylation offers the potential to revert aspects of cancer phenotype with an appropriate therapy (Kalebic T., Ann. Acad. Sci. 293, 2003) .
  • PCR Polymerase chain reaction
  • MSP methylation- specific PCR
  • CpG-RBL2-met CpG-RBL2-met.
  • RBL2 methylation-specific PCR RBL2-MSP
  • RBL2-MSP CpG island methylation test reagent kit
  • Its application serves as biomarkers for early detection of cancer, for risk assessment and for predicting response to therapy.
  • CpG-RBL2-met a CpG island sequence of RBL2 gene sensitive to methylation
  • SEQ ID N0.1 a CpG island sequence of RBL2 gene sensitive to methylation
  • the sequence of CpG-RBL2-met spans from -414bp to +633bp.
  • the sequence of CpG-RBL2-met contains the promoter region immediately 5' -flanking to the transcription start site (ATG) , the exon 1 and the intron 1, and it can be partially or totally methylated in tumor samples.
  • ATG transcription start site
  • the complete CpG island has been divided into three regions.
  • a first region spans from - lOObp to +177bp
  • second region spans from +164bp to 302bp
  • a third region spans from +282bp to +411bp.
  • primers For each region two pairs of primers are provided which amplify the methylated (M1,M2,M3) and unmethylated (Ul, U2,U3).
  • One set of primers has been designed to amplify unmodified DNA (WT) .
  • Estl/2 and Nestl/2 are provided, Estl/2 and Nestl/2, to detect the methylation status of RBL2 gene.
  • Estl and Est2 primers amplify a CpG reach region spans from -378bp to +619bp.
  • Nestl and Nest2 are inside of this region and recognize a region spans from -263bp to +370bp.
  • Both Est and Nest primers are designed to amplify unmethylated modified DNA, therefore all cytosine are converted in uracil. In this case the existence of methylated CG di-nucleotides which are maintained unmodified do not allow the PCR amplification.
  • the invention provides also the method for detecting specific exon 1 hot spot mutations of RBL2 gene identified as tumor-biomarkers, referred to herein as RBL2-exonl-mut, within the DNA samples.
  • RBL2-exonl-mut specific exon 1 hot spot mutations of RBL2 gene identified as tumor-biomarkers
  • Exonl-R has been designed to amplify exonl of RBL2 gene by PCR and the PCR-product sequences can be matched with two GeneBank sequences (accession number X76061 and S67171).
  • the GeneBank sequence X76061 derived from human placenta DNA and is considered wild type while S67171 derived from HeIa human tumor cell line.
  • Mutations at nucleotides 178 and 259 are referred as tumor biomarkers since they are present in tumor samples but not in normal tissues. Mutations found at nucleotide 178 (TCT ⁇ CCT) and
  • Figure 1 shows a sequence of RBL2 retinoblastoma- like 2 promoter, exon 1 and intron 1 within which the CpG island sensitive to methylation is located. Kerl, SpI and MyoD sites are indicated. The position of primers (Est and Nest as well as those used to amplify region 1, 2 and 3) for Methylation specific PCR (MSP) test are indicated. The entire CpG island spans from -378bp to +619bp. The CpG island referred to promoter sequence spans from -378bp to +0; the exon 1 spans from +0 to +240 and the CpG reach region inside intron lspans from +241bp to +619bp.
  • MSP Methylation specific PCR
  • FIG. 2 shows primers for MSP analysis of RBL2 retinoblastoma-like 2 CpG-enriched regions. The position of amplified region, name and sequences of primers, annealing temperature and PCR product size are indicated.
  • Exon 1 primers to identify hot spot mutations at codon 37 and 64 of RBL2 retinoblastoma-like 2 gene recognized as tumor biomarkers The position of amplified region, name and sequences of primers, annealing temperature and PCR product size are indicated.
  • CpG islands which are potential methylation sites, are often found near the promoter of widely expressed genes and typically extend into the first exon . CpG islands can also occur downstream from transcription start sites and are un-methylated in normal cells, although they seem to be preferential targets for de novo methylation in human cancer.
  • the analysis of changes in promoter region methylation serves as a means to inactivate genes in cancer. Understanding these changes may suggest novel pathways altered in cancer which may be view as new targets for drug therapy, may give prognostic information or help to determine a patients response to therapy, and may provide sensitive molecular markers for early detection of cancer.
  • CMOS complementary metal-oxide-semiconductor
  • genomic DNA sequencing Although time consuming and labor intensive, offers a more universal detection method (Yuanxiang Z. et al., BioTechniques 22, 1997) .
  • MSP is now an established technology for the monitoring of abnormal gene methylation in selected gene sequences (Herman, J. G. et al., PNAS USA 93, 1996) .
  • Methylation specific PCR is a simple and sensitive method for the detection of methylation changes in cancer. It allows to identify new genes which are silenced in cancer. With this method it is possible to examine methylation events in pre-invasive lesions associated with solid tumors allowing to understand tumor development and progression and to develop these findings into early detection markers.
  • the assay is based on the DNA sequence differences between methyalted and unmethylated DNA after bisulfite modification by DNA modification Kit (CpGenomeTM, Serologicals® Corporation, Norcross,GA,USA) . By using specific software it is possible to design primers discriminating between methylated, unmethylated and unmodified DNA. PCR reaction performed on bisulfite modified DNA by using primers which recognize either methylated or unmethylated DNA make possible to asses the methylation status of samples
  • Tumorigenesis in humans is the result of multiple processes reflecting genetic and epigenetic alterations that progressively push normal cells towards highly malignant derivatives.
  • Several human tumors involve loss of heterozygosity of tumor suppressor genes and it is now well accepted that mutations or epigenetic silencing of these genes play a key role in malignant transformation.
  • the change in gene expression profiles evident in cancer cells depends on alterations at genetic (i.e., mutations) as well as epigenetic (i.e., transcriptional inactivation due to methylation level (Knudson A. G., Am. J.Med. Genet . 111, 2002; Issa J. P., Crit . Rev. Oncol . Hematol . 32, 1999).
  • tumor suppressor genes can be inactivated by both genetic and epigenetic events thus favoring tumorigenesis .
  • the tumor suppressor Retinoblastoma family genes (RBl, RBL2 and plO7) are able to control cell cycle, apoptosis, differentiation and angiogenesis during normal development. Mutations or functional inactivation of these genes have been found in several tumors and their re- expression have been shown to suppress neoplastic growth. Loss of expression of these tumor suppressor genes could be often due to epigenetic events, which determine gene silencing. Recent studies provided direct evidence that loss of pRbl/plO5 function leads to genome instability and predispose to cancer by increasing DNA mutation rate (Zheng L. and Lee W. H., Adv. Cancer Res. 85, 2002).
  • genomic instability is tightly linked to deregulation of DNA methylation pattern and partial or total gene silencing may be achieved by this epigenetic mechanism (Sugimura T. and Ushijima T. Mutat. Res. 462, 2000).
  • RBL2 retinoblastoma- like 2 gene is impaired in many tumors and often its loss of expression correlates with low apoptotic index suggesting that this gene could be mutated or functionally inactivated.
  • Some mutations impairing its function have been found in specific tumors (Cinti C. et al., Cancer Res. 60, 2000) .
  • no significant mutations are found even if RBL2 retinoblastoma-like 2 gene expression was lost, thus suggesting that other events, such as epigenetic gene silencing, may occur in these tumors.
  • RBL2 mutational analysis in familial and sporadic retinoblastoma, in endometrial carcinoma and in several tumor cell lines. The analysis has been also performed in normal tissues and in blood health donor samples in the attempt of correlate RBL2 retinoblastoma-like 2 gene expression level with gene mutational status. Moreover, we analyzed RBL2 retinoblastoma-like 2 promoter methylation pattern to investigate the relative weight of genetic and epigenetic events in determining the alteration of RBL2 retinoblastoma-like 2 gene expression level.
  • T-limphoblastoid leukemia CCRF-CEM, MoIt-I and Jurkat
  • B-limphoblastoid leukemia Daudi
  • chronic myeloid leukemia K562
  • breast carcinoma SK-Br3 and MCF- 7
  • non small cell lung cancer H23
  • retinoblastoma Weri-Rbl
  • colon cancer HT29
  • Cell lines were purchased from the American Type Culture Collection (Rockinville, MD) . The cells were cultured in DMEM or RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine. DNA extraction protocols
  • the CapsureTM transfer film carrier is placed directly onto a standard microcentrifuge tube containing digestion buffer (50 ⁇ l buffer containing 0.04% Proteinase K, 1OmM Tris-HCL [pH 8.0], ImM EDTA, and 1% Tween-20).
  • digestion buffer 50 ⁇ l buffer containing 0.04% Proteinase K, 1OmM Tris-HCL [pH 8.0], ImM EDTA, and 1% Tween-20.
  • the tube is pre- heated upright in a 37 0 C oven for 5 minutes and then placed upside down so that the digestion buffer contacted the tissue on the cap.
  • the samples are incubated overnight at 37 0 C, centrifuged for 5 minutes and the cap is removed.
  • the samples are heated to 95 0 C for 8 minutes to inactivate the Proteinase K and used directly as template for PCR reactions .
  • the PCR reaction mix (50 ⁇ l) contained genomic DNA at the final concentration of 4ng/ ⁇ l, 0.2mM of each of the four deoxynucleotide triphosphates, 2U of Taq I polymerase (Promega, USA) and primers (Exon 1-F: 5'-CCT CAC CTC ACC TGA GGT-3 1 ; Exon 1- R: 5'-ACC GGT TCA CAC CAA CTA GG-3'; product size: 329bp) at the final concentration of 0.4 ⁇ M each.
  • DNA is purified using the QUIAquick gel purification kit (Qiagen, Santa Clarita, CA) and used for automated DNA sequencing.
  • the sequence analysis of the purified PCR products is performed in both the sense and antisense directions and is carried out by automated DNA-sequencing using dideoxy-terminator reaction chemistry (Big-dye® terminator kit, Applied Biosystem, Foster City, CA, U.S.A.) for sequence analysis on the Applied Biosystem model 373A DNA Sequencer.
  • T- limphoblastoid leukemia CCRF-CEM, MoIt-I and Jurkat
  • B- limphoblastoid leukemia Daudi
  • chronic myeloid leukemia K562
  • breast carcinoma SK-Br3 and MCF-7
  • non small cell lung cancer H23
  • retinoblastoma Weri-Rbl
  • colon cancer HT29
  • these missense mutations occur in a region, the exon 1, rich in CpG islands potentially target for epigenetic events supporting the hypothesis that these mutations could influence the susceptibility of the CpG region to methylation and thus determining the loss of RBL2 protein expression.
  • No-small-cell-lung cancer (H23) and retinoblastoma (Weri-Rbl) cell lines, and primary tumors from patients with familiar and sporadic retinoblastoma and endometrial carcinoma have been analyzed.
  • As control normal retina and endometrius tissues from patients affected by non- tumoral pathologies and in health donors blood samples have been tested.
  • Cell lines were purchased from the American Type Culture Collection (Rockinville, MD) . The cells were cultured in DMEM or RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine . Case selection and processing of tissue for histological evaluation
  • the histopathological type of these cancers was 34 endometrioid cancer.
  • 12 samples of normal endometrium were collected from 12 patients undergoing surgery for benign gynecologic disease. Tissues were cut and fixed in a buffered 4% aqueous formaldehyde solution, pH 7.4.
  • 4 ⁇ m-thick sections were obtained from representative paraffin wax blocks and stained with hemalum and eosin, Giemsa, periodic acid-Schiff (PAS), Gomori's silver impregnation, and Feulgen.
  • Genomic DNAs were extracted from frozen samples, tumor cell line and 15 health donors blood samples with phenol/chloroform using a procedures described in Maniatis
  • Archived paraffin-fixed normal human retina and retinoblastoma samples are identified on H&E-stained sections and isolated by laser capture microdissection (Arcturus PixCell IITM MWG-BIOTECH, Florence, Italy) .
  • the selected samples adhered to a CapsureTM transfer film.
  • the CapsureTM transfer film carrier was placed directly onto a standard microcentrifuge tube containing digestion buffer (50 ⁇ l buffer containing 0.04% Proteinase K, 1OmM Tris-HCL [pH 8.0], ImM EDTA, and 1% Tween-20).
  • the tube was pre ⁇ heated upright in a 37 0 C oven for 5 minutes and then placed upside down so that the digestion buffer contacted the tissue on the cap.
  • the samples were incubated overnight at 37 0 C, centrifuged for 5 minutes and the cap was removed.
  • the samples were heated to 95 0 C for 8 minutes to inactivate the Proteinase K and used directly as template for bisulphite-modification
  • DNAs extracted from frozen tissue, paraffin-fixed tissue and cell lines were treated with sodium bisulfite and analyzed unsing methylation specific PCR (MSP-PCR) as described by Herman et al., 1996 as well as using "CpGenomeTM DNA Modification Kit” (cod.S7820, Serologicals® Corporation, Norcross, GA, USA) . Briefly, 3-5 micrograms of genomic DNA is dissolved in 20 ⁇ l of deionised water. Samples are first exposed to DNA denaturation to its single stranded form using mild heat (50 0 C) at an alkaline pH (0.3M NaOH solution).
  • DNA is then bound to a micro-particulate carrier and is desalted by repeated centrifugation and resuspension in 70% EtOH.
  • the conversion to uracil is completed by alkaline desulfonation (incubation at RT for 10 minutes in a 20 mM NaOH/90% EtOH solution) and desalting is repeated in 90% EtOH.
  • the DNA is finally eluted from the carrier by heating in TE IX Buffer.
  • CpG WareTM Primer Design Software is able to design primers discriminating between methylated, unmethylated and unmodified DNA. PCR reaction performed on bisulfite modified DNA reveal the methylation status of samples .
  • Amplifications were carried out in a 50 ⁇ l volume containing IX PCR buffer (EuroClone, Devon, UK), 2.5mM MgC12, dNTPs (each at 25 mM) , 300ng of each primers, 1 unit of EuroTaq (EuroClone) .
  • Amplification was carried out in a Perkin Elmer model 9600 thermocycler at 95°C for 5 min, followed by 35 cycles (1 min at 95°C, 1 min at the annealing temperature, and 1 min at 72 0 C) followed by a final 7-min extension at 72°C.
  • Each PCR products (15ul) was directly loaded onto 2% agarose gels, stained with ethidium bromide, and directly visualized under UV illumination.
  • Estl/2 and Nestl/2 are designed to detect the methylation status of RBL2 gene.
  • Both Est and Nest primers are designed to amplify un- methylated modified DNA, therefore all cytosine are converted in uracil. In this case the existence of methylated CG di-nucleotides, which are maintained unmodified, do not allow the PCR amplification.
  • the same sample is amplified using Est and Nest primers.
  • the first pair of primers (Estl and Est2) amplified the entire CpG locus (from -378bp to +619bp) .
  • the PCR products amplified with Estl and Est2 are processed for a 2nd round of PCR. Amplification were carried out in a 50 ⁇ l volume containing 5 ⁇ l of 1st round PCR product, IX PCR buffer, 2.5mM MgC12, dNTPs (each at 25 mM) , 300ng of Nestl and Nest2 primers, 1 unit of EuroTaq (EuroClone) .
  • Amplification is performed by pre-heating step (95°C for 5 min) followed by 35 cycles (1 min at 95°C, 1 min at the annealing temperature, and 1 min at 72°C) followed by a final 7-min extension at 72°C.
  • PCR products (15ul) were directly loaded onto 2% agarose gels, stained with ethidium bromide, and directly visualized under UV illumination.
  • the regions 1 (-95bp to +177bp) and 3 (+287bp to +411bp) were found methylated in No-small-cell-lung cancer (H23) cells and the only region 3 in retinoblastoma (Weri-Rbl) cell line. In both cell lines, the region 2 (+164bp to 302bp) is not methylated.
  • the MSP . analysis has also been performed in endometrial carcinoma and retinoblastoma primary tumors and the results have been correlated with the immunohistochemical analysis. It has been show that when RBL2 expression level is normal (as demonstrated by histological evaluation) all the three examined regions were un-methylated. On the contrary, loss of the expression corresponded to methylation of the regions 1, 2 and 3, while down-regulation was correlated to methylation of region 3 alone.
  • loss of expression is correlated with double homozygous mutation
  • weak expression coincides with the presence of 178 homozygous and 259 heterozygous mutations while when both mutations are heterozygous the expression level is normal.
  • These mutations occurring in the exon 1 region, rich in CpG dinucleotides, could be the hit event that predisposes RBL2 retinoblastoma-like 2 gene to epigenetic changes leading to the silencing of the gene in cancer.
  • Exon 1 is a CpG enriched region and, therefore, it is susceptible to be improperly methylated.
  • Screening of methylation status in RB2/pl30 gene promoter shows that a 178 (TCT ⁇ CCT) homozygous mutation in Exon 1 correlates with hypermethylation at one site determining a reduction in protein expression level. Actually this mutation alters susceptibility to methylation of a region (M3) bridging Exon 1 and Intron 1 thus allowing transcription even if it is impaired.
  • a 259 (CCC->GCC) homozygous mutation adds to the other one, promoter as well Exon 1 and Intron 1 regions becomes aberrantly methylated and transcription is abolished.

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Abstract

La présente invention concerne l'utilisation d'une séquence génique humaine, un îlot de CpG spécifique du gène 2 de type rétinoblastome (pl30), ainsi que la conception d'amorces, fondée sur le principe de base du mésappariement, pour constituer une trousse de réactifs destinés à des tests de méthylation d'îlots de CpG permettant le diagnostic d'une tumeur et le criblage d'un médicament de déméthylation. La trousse de réactifs inclut des amorces spécifiques du système réactionnel MSP-PCR. L'invention concerne également la production d'un îlot de CpG entier, sensible à la méthylation. Elle concerne de plus des amorces spécifiques permettant la détection de mutations de « points chauds » au niveau de l'exon 1, identifiées en tant que biomarqueurs tumoraux, et elle implique la réaction de PCR. Trois régions du gène 2 de type rétinoblastome (pl30), riches en dinucléotides CpG, la région promoteur flanquant immédiatement en 5' le site d'initiation de la transcription (ATG), l'exon 1 et l'intron 1, représentent des cibles pour des événements épigénétiques dans des tumeurs. Deux mutations spécifiques de l'exon 1, au sein d'une région de CpG sensible à la méthylation, sont présentes dans différents hystotypes de cancer et représentent un marqueur spécifique de la tumeur. L'analyse des modèles de méthylation dans les régions du gène 2 de type rétinoblastome (pl30), riches en dinucléotide CpG, au moyen d'une PCR spécifique de la méthylation (MSP), et l'analyse mutationnelle de l'exon 1 conduisent à des procédés permettant de poser un diagnostic et un pronostic et d'élaborer une thérapie de ciblage chez des patients cancéreux.
PCT/EP2006/005019 2006-05-26 2006-05-26 Tests destinés à la détection de mutations de points chauds et de la méthylation du gène 2 de type rétinoblastome (rbl2) utilisées comme marqueurs diagnostiques et pronostiques de tumeurs Ceased WO2007137597A1 (fr)

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Cited By (1)

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CN119619524A (zh) * 2024-12-10 2025-03-14 复旦大学附属眼耳鼻喉科医院 特异性结合s-100b蛋白的物质在制备急性视网膜坏死诊断产品或预后产品中的用途

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WO2002077272A2 (fr) * 2001-03-26 2002-10-03 Epigenomics Ag Procedes et acides nucleiques pour l'analyse des troubles de la proliferation des cellules hematopoietiques
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