WO2006111586A2 - Procede permettant de determiner in vitro le degre de methylation du promoteur de line-1 - Google Patents

Procede permettant de determiner in vitro le degre de methylation du promoteur de line-1 Download PDF

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WO2006111586A2
WO2006111586A2 PCT/ES2005/000205 ES2005000205W WO2006111586A2 WO 2006111586 A2 WO2006111586 A2 WO 2006111586A2 ES 2005000205 W ES2005000205 W ES 2005000205W WO 2006111586 A2 WO2006111586 A2 WO 2006111586A2
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line
methylation
degree
promoter
seq
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PCT/ES2005/000205
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Spanish (es)
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WO2006111586A3 (fr
WO2006111586B1 (fr
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Xabier Aguirre Ena
GÓMEZ José ROMÁN
Antonio JIMÉNEZ VELASCO
Felipe Prosper Cardoso
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Proyecto De Biomedicina Cima, S.L.
Asociación Medica E Investigación (A.M.I.)
Fundación Imabis
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Publication of WO2006111586A3 publication Critical patent/WO2006111586A3/fr
Publication of WO2006111586B1 publication Critical patent/WO2006111586B1/fr

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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
    • C12Q1/6876—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
    • 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
    • C12Q2600/106—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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention falls within the scope of the methods of detecting the degree of methylation of the LINE-I promoter.
  • Chronic Myeloid Leukemia is a malignant disease of hematopoietic stem cells, which has an incidence of 1.2: 100,000 inhabitants and years. It has three distinct phases characterized both at the phenotypic level and from the point of view of the treatment and the prognosis of the disease (chronic phase -3 to 4 years- accelerated phase -1 to 2 years- and finally a blastic phase -6 to 12 months - in which the disease is refractory to treatment).
  • the transition from a Chronic Phase to a Blastic Crisis is characterized by genomic instability in which an accumulation of molecular and chromosomal alterations is observed, but the mechanism by which this genomic instability occurs is still unknown. The possibility of determining mechanisms involved in genomic instability would favor the development of new therapeutic and diagnostic strategies.
  • Imatinib a drug directed specifically against the molecular target of the disease called Imatinib has recently been discovered, capable of inhibiting the BCR-ABL oncogene responsible for the disease.
  • Philadelphia chromosome is the specific marker of the disease and appears as a consequence of a reciprocal translocation between chromosomes 9 and 22 resulting in an abnormal protein with tyrosine activity
  • DNA methylation plays a very important role in maintaining genomic stability and that in particular the hypomethylation of DNA in tumor cells is associated with instability. DNA methylation changes can
  • REPLACEMENT SHEET (Rule 23) occur both in regions close to promoters and first exons of different genes (as a mechanism to control their transcription) and in repetitive regions of the genome rich in CpG dinucleotides. Abnormal (increased) methylation of said promoters results in the silencing of their expression.
  • retrotransposons In addition to the specific regulation of genes through the methylation of their promoters, in the human genome up to 20% of the sequences are formed by a series of repetitive elements called retrotransposons and that under normal conditions are hypermethylated so that they are not expressed. These retrotransposons are characterized by their ability to transcribe to RNA, retrotranscribe to DNA and that that DNA is inserted into a new region of genomic DNA contributing to the regulation of some genes, and in turn, facilitating alterations at the gene level.
  • One of the most frequent retrotransposons is LINE-I. When retrotrasposons jump from one region of the genome and are inserted into another, they can contribute to inducing genome alterations, especially when they are not silenced by hypermethylation of their promoter regions. It is unknown, however, which genes are affected as a result of LINE-I hypomethylation.
  • DNA methylation changes can occur both in regions close to promoters and first exons of different genes and in repetitive regions of the genome rich in CpG dinucleotides. In regions close to the promoters and first exons of different genes, the alteration of the methylation that usually occurs is a hypermethylation that normally leads to a decrease or loss of expression of those genes.
  • hypermethylation of different genes plays a very important role in the progression and response of different types of tumors (Román-Gómez et
  • REPLACEMENT SHEET (Rule 2S) to the. Promoter hypermethylation of cancer related genes is a strong independent prognostic factor in Acute Lymphoblastic Leukemia; Blood 2004; 104: 2492-2498).
  • the LINE-I 1 elements are one of the most important elements. These elements are usually methylated, in this case their hypomethylation is the mechanism of alteration. Different studies have described that these elements can be inserted within the regions of the genes, being able to control and modify the normal transcription of these genes (Druker et al. Complex patterns of transcription at the insertion site of a retrotransposon in the mouse. Nucleic Acids Research ; 2004; 32: 5800-5808; Han et al. Transcriptional disruption by the Ll retrotransposon and implications for mammalian transcriptomes. Nature, - 2004; 429: 268-274).
  • LINE-I is hypomethylated and in the case of prostate carcinoma it has been observed that this hypomethylation may be involved in the progression of the disease (Chalitchagorn et al. Distinctive pattern of LINE-I methylation level in normal tissues and the association with carcinogenesis; Oncogene; 2004; 23: 8841-8846; Florl et al. Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-I hypomethylation; British Journal of Cancer; 2004; 91: 985- 994). But so far it was not known what role hypomethylation of LINE-I plays in predicting the prognosis of the disease.
  • Figure 1 Methylation status of the LINE-I promoter in patients and CML cell lines.
  • REPLACEMENT SHEET (Rule 26) unmethylated sequence; M: methylated sequence.
  • hypermethylation of the LINE-I promoter is observed (lack of amplification of the non-methylated sequence) while in the samples of patients with CML, different degrees of hypotnetylation of the LINE-I promoter are observed (amplification of both the methylated sequence as of the unmethylated sequence).
  • Figure 2 Distribution of the disease-free survival curve (PFS) by the Kaplan and Meier method in patients with CML according to the methylation status of the LINE-I promoter.
  • PFS disease-free survival curve
  • Figure 3 Effect of hypomethylation of LINE-I on the expression of ORF-I and c-MET.
  • UM unmethylated sequence
  • M methylated sequence ' .
  • the present invention relates to a method for in vitro determination of the degree of methylation of the LINE-I promoter, characterized in that it comprises amplifying genomic DNA by means of a pair of primers.
  • REPLACEMENT SHEET (Rule 26) specific amplification of unmethylated sequences and a pair of amplification primers specific for methylated sequences, where both pairs:
  • said primers preferably have a size of between 15 and 25 nucleotides.
  • At least one of the amplification primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ.
  • primers specific for methylated sequences possess the sequences corresponding to SEQ. ID. NO: 1 and 2.
  • primers specific for non-methylated sequences have sequences corresponding to SEQ. ID. NO: 3 and 4.
  • the method for in vitro determination of the degree of methylation of the LINE-I promoter is a methylation sensitive PCR. And, in a more preferred embodiment, said method is a methylation sensitive PCR by real time quantification.
  • the process for in vitro determination of the degree of methylation of the LINE-I promoter is
  • SHEET LEAVE (Rule 26) characterized in that a degree of methylation less than 80% is related to the presence of leukemia.
  • the leukemia is related to a degree of methylation of the LINE-I promoter of less than 70%.
  • said degree of methylation related to the presence of leukemia is less than 50%.
  • said leukemia is acute leukemia or chronic leukemia.
  • said leukemia is selected from: a chronic myeloid leukemia, an acute myeloid leukemia, an acute lymphocytic leukemia or a chronic lymphocytic leukemia.
  • the in vitro determination of the degree of methylation of the LINE-I promoter makes it possible to judge the evolution of said leukemia.
  • said determination may also allow to evaluate the evolution of said leukemia in response to various treatments.
  • the present invention relates to an oligonucleotide selected from SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4.
  • the present invention relates to a kit for determining the degree of methylation of the LINE-I 7 promoter characterized in that it comprises a pair of amplification primers specific for unmethylated sequences and a pair of amplification primers specific for methylated sequences .
  • said kit for determining the degree of methylation of the LINE-I promoter is characterized in that both pairs of primers:
  • REPLACEMENT SHEET (Rule 28) - at least one of them selectively hybridizes with a sequence comprised between nucleotides 100 and 150, or between nucleotides 240 and 290 of said promoter consensus sequence.
  • said pair of amplification primers specific to methylated sequences are characterized in that at least one of the primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4.
  • the pair of amplification primers specific for methylated sequences are selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4.
  • kits for determining the degree of methylation of the LINE-I promoter it comprises appropriate reagents for performing a methylation sensitive PCR method.
  • the present invention relates to a method of diagnosis of leukemia, by determining the presence of hypomethylation of the LINE-I promoter, so that a hypomethylation is considered indicative of leukemia, when its levels are below 80 %.
  • said hypomethylation is less than 70%, and in a more preferred embodiment, said hypomethylation is less than 50%.
  • said method allows to predict the evolution of said leukemia, and / or determine the response of said leukemia to various treatments.
  • the analysis of the methylation status of the LINE-I promoter was carried out in 140 patients affected by chronic myeloid leukemia (CML) diagnosed between 1982 and 2003.
  • CML chronic myeloid leukemia
  • bone marrow was obtained from each patient in the time of diagnosis (before receiving any treatment) and healthy bone marrow donors. In all cases, the bone marrow sample was obtained after informed consent. From the bone marrow sample, the population of mononucleated cells was obtained after sedimentation in a Ficoll-Hypaque gradient. The bone marrow sample at diagnosis was obtained in all patients, while in 47 both the sample was obtained at diagnosis and the sample at the time of the blast crisis (34 in myeloid blast crisis and 13 in lymphoid blast crisis).
  • Blastic crisis was defined as the presence of 30% blasts in peripheral blood or bone marrow.
  • the DNA for subsequent analysis of the methylation status of the LINE-I promoter was obtained from each bone marrow sample by using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany).
  • the quantitative MSP technique was used to analyze the methylation status of the LINE-I promoter.
  • the MSP (Methylation Specific PCR) technique is based on a first treatment of the DNA sample with sodium bisulfite, which modifies unmethylated cytosines to uracil, while cytosines
  • REPLACEMENT SHEET (Rule 26) methylated do not undergo any change after this treatment.
  • 1 ⁇ g of DNA from each bone marrow sample of patients with CML was treated and modified using the CpGenomic TM DNA modification Kit (Intergen Company, Purchase, NY).
  • the protocol of this modification kit consists briefly of the following:
  • DNA Modification Reagent II (provided by the kit) and mix.
  • Preparation of DNA Modification Reagent II First add 1 ⁇ l of ⁇ -mercaptoethanol to 20 ml of deionized water. For each sample to be modified, add 750 ⁇ l of the ⁇ -mercaptoethanol solution to 1.35 g of the DNA Modification Reagent II. - Incubate for 10 minutes at room temperature.
  • REPLACEMENT SHEET (Rule 26) - Add 1 ml of 70% ethanol, vortex, centrifuge 10 seconds 5000 xg and remove the supernatant. Repeat this process 3 times.
  • REPLACEMENT SHEET (Rule 26) After modification of the DNA by means of sodium bisulfite, the MSP was performed for the analysis of the methylation status of the LINE-I promoter. This MSP consists of two PCRs, one performed with specific primers for amplification of the LINE-I promoter in the methylated state and a second PCR performed using specific primers for amplification of the LINE-I promoter in the non-methylated state.
  • Quantitative MSP was performed using a LightCycler (Roche Molecular Biochemicals) with the ability to discriminate three fluorescent colors using 1 ⁇ l of DNA modified with sodium bisulfite in a final reaction volume of 10 ⁇ l with 0.4 ⁇ mol / 1 of each primer, 3 , 5 mmol / 1 Mg 2+ and 1 ⁇ l of the 1Ox LightCycler FastSatr DNA Master SYBR Green I (Roche Molecular Biochemicals).
  • the amplification of the LINE-I promoter in the methylated state was carried out by using the primers: sense, 5 '-GTCGAATAGGAATAGTTTCGG-3'; antisense, 5 '-ACTCCCTAACCCCTTACGCT-3'; and amplification of the LINE-I promoter in a non-methylated state by using the primers: sense, 5 '-GTTGAATAGGAATAGTTTTGGTTT-3 •; antisense, 5'- ACTCCCTAACCCCTTACACTT-3 '.
  • the amplification conditions consisted of: denaturation program consisting of a cycle _ 95 ° C 10 minutes; amplification program, consisting of 45 cycles at 95 ° C 1Os, 65 ° C 1Os and 72 ° C 1Os, • melting program, consisting of a cycle at 95 ° C Os, 40 0 C 6Os and 90 0 C for 6Os, • and a cycle cooling program at 40 0 C 6Os.
  • the temperature transition was 20 ° C / s, except in the melting program that was 0.4 ° C / s between 40 0 C and 90 0 C.
  • Normalised ratio (N L1 ) (E target ) ⁇ Cp target (contro1 " sample) ⁇ Cp ref (control - sample) normalised ratio: normalized ratio, target: target, control: control, sample: sample.
  • the bone marrow of 50 healthy individuals was analyzed.
  • the LINE-I promoter was methylated in healthy subjects, but in some cases amplification of both the methylated and non-methylated sequence was obtained. Due to these data, the sample of healthy individual that presented the smallest difference in amplification between the methylated and nonmethylated sequences was taken as a control, considering that in this case the LINE-I promoter was 100% methylated. Based on this prerequisite, in the analysis of the 50 samples of healthy individuals, values between 100% and 231% were obtained, the average being 160% ⁇ 45%. Thanks to this data, the value of 70% (determined as the mean minus 2 SD) was chosen as the cut-off point, defining
  • REPLACEMENT SHEET (Rule 26) ratios equal to or less than this value as hypomethylation of the LINE-I promoter.
  • This program allows to design primers for the analysis of methylation by means of MSP-PCR, analysis of methylation by restriction enzymes sensitive to methylation and sequencing analysis after treatment with sodium bisulfite.
  • the parameters that were taken into account were the following:
  • primers were designed on this region. These primers had to comply with the following:
  • Example 3 Chronic myeloid leukemia. Degree of methylation of LINE-I.
  • LINE-I methylation was analyzed in 50 samples of normal bone marrow. As expected, the LINE-I promoter sequences were strongly methylated, which is manifested by the amplification of the methylated sequences, according to the method described in the previous examples, with the absence of amplification of non-amplified sequences. in most samples (72%).
  • the analysis of the methylation of the LINE-I promoter in samples of patients with chronic myeloid leukemia resulted in the amplification of unmethylated promoter sequences, said hypomethylation being more pronounced in samples from patients in blast crisis than in patients in phase chronic (Figure IA).
  • Example 4 Chronic myeloid leukemia. Determination of cut-off or reference values
  • the bone marrow of 50 healthy individuals was analyzed.
  • the LINE-I promoter was methylated in healthy subjects, but in some cases amplification of both the methylated and non-methylated sequence was obtained. Due to these data, the sample of healthy individual that presented the smallest difference in amplification between the methylated and nonmethylated sequences was taken as a control, considering that in this case the LINE-I promoter was 100% methylated. Based on this prerequisite, in the analysis of the 50 samples of healthy individuals, values between 100% and 231% were obtained, the average being 160% + 45%. The value of 70% (determined as the mean minus 2 SD) was chosen as the cut-off point, defining ratios equal to or less than this value as hypomethylation of the LINE-I promoter.
  • Example 5 Chronic myeloid leukemia. Analysis of patients diagnosed with CML.
  • Example 6 Chronic myeloid leukemia. LINE-I hypomethylation is associated with the activation of c-MET antisense transcription.
  • LINE-I has two transcription regulatory regions located in the 5 'untranslated region: • an internal or sense promoter that directs the complete LINE-I transcription (ORF-I), and an antisense promoter
  • LINE-I could affect the expression of c-MET. It was observed that c-MET expression was significantly higher in those patients with chronic myeloid leukemia who had the hypomethylated LINE-I promoter. Similarly, c-MET overexpression was detected in 61% of patients with chronic myeloid leukemia with
  • said chronic phase patient with LINE-I hypomethylation has an increased risk of resistance to current reference treatments (interferon or Imatinib);

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Abstract

L'invention concerne un procédé permettant de déterminer in vitro le degré de méthylation du promoteur de LINE-1. Ce procédé est caractérisé en ce qu'il comprend l'amplification de l'ADN génomique à l'aide des amorces d'amplification spécifiques.
PCT/ES2005/000205 2005-04-20 2005-04-20 Procede permettant de determiner in vitro le degre de methylation du promoteur de line-1 WO2006111586A2 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP2210954A1 (fr) * 2009-01-22 2010-07-28 Heinrich-Heine-Universität Düsseldorf Détermination du niveau de méthylation de l'ADN
CN114182005A (zh) * 2021-10-29 2022-03-15 上海普然生物科技有限公司 一种用于line-1甲基化检测的检测试剂盒及其检测方法和应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2210954A1 (fr) * 2009-01-22 2010-07-28 Heinrich-Heine-Universität Düsseldorf Détermination du niveau de méthylation de l'ADN
WO2010084154A3 (fr) * 2009-01-22 2010-12-02 Heinrich-Heine-Universität Düsseldorf Détermination du degré de méthylation de l'adn
CN102356160A (zh) * 2009-01-22 2012-02-15 杜塞尔多夫海因里希·海涅大学 Dna甲基化水平的测定
US9850528B2 (en) 2009-01-22 2017-12-26 Heinrich-Heine-Universität Düsseldorf Determination of the normalized degree of DNA methylation
CN114182005A (zh) * 2021-10-29 2022-03-15 上海普然生物科技有限公司 一种用于line-1甲基化检测的检测试剂盒及其检测方法和应用

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