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WO2003035907A2 - Methode et moyens d'identification precoce de risques de decompensation cardiaque - Google Patents

Methode et moyens d'identification precoce de risques de decompensation cardiaque Download PDF

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Publication number
WO2003035907A2
WO2003035907A2 PCT/IB2002/004351 IB0204351W WO03035907A2 WO 2003035907 A2 WO2003035907 A2 WO 2003035907A2 IB 0204351 W IB0204351 W IB 0204351W WO 03035907 A2 WO03035907 A2 WO 03035907A2
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WO
WIPO (PCT)
Prior art keywords
genes
microarray
cdna
class
series
Prior art date
Application number
PCT/IB2002/004351
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English (en)
Other versions
WO2003035907A3 (fr
Inventor
Carlo Ventura
Federico Giudiceandrea
Giandomenico Testi
Original Assignee
Carlo Ventura
Federico Giudiceandrea
Giandomenico Testi
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
Application filed by Carlo Ventura, Federico Giudiceandrea, Giandomenico Testi filed Critical Carlo Ventura
Publication of WO2003035907A2 publication Critical patent/WO2003035907A2/fr
Publication of WO2003035907A3 publication Critical patent/WO2003035907A3/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
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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 relates to a method suitable to identify risks of onset of cardiac decompensation and to a testing system and apparatuses for performing said method.
  • the method is therefore useful for diagnostic purposes and is particularly advantageous for early diagnosis, in the preclinical phase, of these risks.
  • Heart diseases as is known, are a major cause of death.
  • Myocardial infarction the various forms of diabetes and hereditary cardiomyopathies are on the rise as causes of cardiac damage and failure in modern society. Many of these diseases lead to myocardial hypertrophy, which in turn often evolves toward a final stage of cardiac decompensation. Such a transition is a crucial passage in the clinical course of acquired and congenital cardiomyopathies.
  • the aim of the present invention is therefore to provide a method that is capable of preventing the risks of the onset of cardiac decompensation.
  • An object therefore, is to provide a diagnostic method and a computerized process, and the means for performing it, that can identify the risks of development of cardiac decompensation, especially in patients with myocardial hypertrophy.
  • Another object of the invention is to provide a method and means for early preclinical diagnosis of the risks of onset of cardiac decompensation. The inventors have now found that it is possible to predict the risk of evolution toward cardiac decompensation, and therefore its onset, on the basis of the analysis of the gene expression profile in a suitable biological sample of a subject, particularly a subject affected by myocardial hypertrophy.
  • the inventors have provided a method and a system suitable to provide early diagnosis, advantageously in the preclinical phase, of the risks of onset of cardiac decompensation.
  • the invention is based on the discovery, made following gene expression studies in transgenic animals, that myocardial hypertrophy, when it is destined to evolve toward a final stage of cardiac decompensation, leads to the activation of a fetal gene expression program that is typical of cardiogenesis in embryo development but is usually absent in the adult heart. It has in fact now been discovered that genes involved in the orientation of cardiogenesis of embryo stem cells, or genes whose expression is usually limited to the fetal period, are overexpressed in the adult heart during initial phases of hereditary and acquired cardiomyopathies that precede the onset of cardiac decompensation.
  • the present invention provides a method for identifying risks of onset of cardiac decompensation that is characterized in that it comprises the analysis of the gene expression profile of a biological sample in order to identify the expression of genes that are normally active only in the embryonic phase in the myocardial differentiation of stem cells, said genes belonging to at least one of the classes that comprise (A) genes involved in myocardial development and growth (B) genes that trigger apoptotic cell death, and (C) genes involved in the survival mechanisms of myocardial cells.
  • the present invention also provides a semiautomatic computerized process for performing the method mentioned above and a computer-controlled device for performing said method, as
  • the invention consists of a method that allows to correlate a specific gene expression profile with a state of transition of the myocardium toward cardiac decompensation.
  • This method is based on the discovery that during the initial or early phase of myocardial hypertrophy, the o resumption in the adult of a gene expression profile involved in the determination of the cardiac phenotype during the embryonic period reveals a substrate of risk for the transition from hypertrophy to cardiac decompensation.
  • Such a gene expression profile appears during a period that is devoid of symptoms, which anticipates by years the appearance of the clinical symptoms
  • the method according to the mvention is particularly advantageous in allowing early diagnosis of the risks of onset of cardiac decompensation, which can anticipate by years the appearance of clinical symptoms, the only ones that currently allow to diagnose the disease.
  • D genes whose function is currently not fully clarified but whose activation however indicates the risk of transition of the myocardial hypertrophy toward cardiac decompensation.
  • cardiac tissue activates a series of genes that trigger a program of apoptotic cell death (cell suicide) together with another series of genes that mediate cell survival.
  • the normal growth of the myocardium occurs in a tightly controlled equilibrium between the apoptotic mechanisms and the survival-related mechanisms, which coexist with the expression of genes that control the transverse sectional area (volume) of cardiac myocytes (the contractile unit in heart tissue).
  • the transition from compensated hypertrophy to pathological hypertrophy is determined by an imbalance in this gene expression profile, which ultimately leads to the overexpression of the genes that control the growth of the myocardium and of the genes involved in apoptotic cell death.
  • PLC Protein kinase C
  • ⁇ opioid receptor 18.
  • CREB Cyclic AMP Response Element-binding Protein
  • IGF 1 insulin-like growth 20.
  • ANP atrial natriuretic factor 1 peptide
  • TGF- ⁇ Transforming 22.
  • Egr-1 26.
  • ODC Ornithine decarboxylase
  • genes that code for, and express, the following proteins have been identified as genes of class C) involved in the mechanisms related to cell survival:
  • Phosphoinositide-dependent 3-kinase PI3K
  • class A genes in the absence of expression of class B) and C) genes is correlated to a gradual increase in ventricular mass, with a modest risk of evolution toward cardiac decompensation;
  • the risk can be considered minimal if the expression of class A) genes is associated with the expression of class C) genes;
  • a suitable biological sample preferably a
  • the corresponding cDNA is prepared and marked by reverse transcription of the former and amplification thereof, for example by means of the PCR (Polymerase Chain Reaction) technique.
  • the prepared cDNA is marked, for example after its synthesis, by terminal marking or preferably by introducing a suitable marker during said amplification. This can be done by binding the marker to one of the primers or to one of the nucleotides used in the amplification process.
  • marking in the method according to the present invention is of the fluorescent type, which can be detected for example with a scanner, for example a confocal laser scanner.
  • the fluorescent single-filament cDNA thus prepared starting from the biological sample is analyzed to determine the gene expression profile in said sample.
  • the cDNA is applied to a microarray or chip of DNA oligonucleotides, which are complementary to selected sequences of the genes that belong to classes A, B, C and/or D mentioned above, whose expression one seeks to identify in the sample.
  • the microarray or chip of DNA oligonucleotides is prepared by virtue of per se known methods or with the technique of in situ synthesis on a chosen substrate or by applying to the chosen substrate the oligonucleotides in pre-synthesized form. Methods for preparing said microarrays are described for example in US patent 5,143,854 or in European patent 476,014.
  • the substrate used for the array is preferably glass, although other porous or non-porous substrates can be used, such as films, sticks, pearls, filaments, et cetera of materials such as silica, plastic, nylon or nitrocellulose.
  • oligonucleotides that are 20 to 70 bases long, more preferably 40 to 50 bases long, and are complementary to specific and highly preserved sequences of the sought genes of the respective classes A) to D). It is possible to prepare on a substrate a microarray that contains at least one series of oligonucleotides that comprises an oligonucleotide that is complementary to each one of the genes of classes A) to D) being sought.
  • microarrays are preferably used which comprise duplicate or triplicate replications of a full series of said oligonucleotides.
  • a second series or replication, and optionally a third series or a repeat can be identical or not to the first series of oligonucleotides.
  • the marked cDNA applied to the chip contains one or more of the sought genes of classes A) to D), these genes form hybrids with the complementary oligonucleotides in the microarray.
  • the chosen hybridization conditions are such as to allow only the forming of hybrids with oligonucleotide filaments that are fully or highly complementary, without retaining on the chip nucleic acids that are not perfectly complementary with respect to the oligonucleotides of the microarray.
  • temperatures of 40-50 °C in the presence of suitable buffers to maintain the necessary ion strength of the solution are used for this purpose. In these conditions, stable hybrids are formed only with genes that match perfectly the oligonucleotides of the microarray.
  • a computer program capable of processing the measured data by comparing them with a library of previously measured and acquired data, a quantitative evaluation of the intensity and a qualitative evaluation of the position of the fluorescence correlated with each individual gene, and the processing of a global gene expression profile that is preferably correlated with an index of risk of cardiac decompensation development.
  • This correlation is then presented in the form of a diagnostic report that can be organized with various criteria, for example as a global report, as a detailed report of the intensity of expression of individual genes, as a plot of gene expression profiles, total or by classes, and as relevant statistics.
  • the laser scanner detects, by means of measurements of reflected light, the intensity of the fluorescence signal, which is correlated to the quantitative level of activity of that given gene in the myocardial cells of the patient being tested; therefore, the greater the fluorescence, the greater the presence of that particular gene and its expression in the respective cells.
  • the probability of an incipient evolution toward cardiac decompensation depends on the intensity of the expression of each individual gene and on the number of genes clearly expressed, depending on which of the various classes A) to D) they belong to, as explained above.
  • a process for performing a method for identifying the gene expression profile as described above, using a computer program that oversees the execution of all the steps of the process and in particular:
  • the program checks the execution of the steps of extraction of the mRNA from the biological sample, reverse transcription and amplification thereof to cDNA, deposition of the cDNA on the microarray, its hybridization with the complementary ohgomers, and finally the washing of the microarray;
  • the program starts the operation of a scanner to sense the fluorescence on the microarray
  • the program during its operation, can also perform self-updating and calibration of the reference data library on the basis of information related to the clinical data of the patients and of auto-diagnosis of the correct operation of the monitoring process.
  • a computer-controlled device for performing the method for identifying the gene expression profile according to the invention, said device comprising:
  • - means for treating the biological sample in order to prepare the marked cDNA said means being arranged on a frame that can perform an to advancement that is controlled by the computerized program.
  • means which are per se known, comprise for example containers, means for quantifying the extracted RNA, for example a spectrophotometric device, automated means for drawing preset microvolumes that correspond to preset quantities of total RNA that are ideal for subsequent reverse transcription,
  • the scanner is provided with cartridges for accommodating and positioning the chips of DNA ohgomers for measuring the fluorescence on said chips.
  • the example that follows illustrates a specific protocol that can be applied in the execution of the steps of cDNA synthesis and hybridization according to the method of the present invention.
  • the reaction volume is 50 ⁇ l
  • oligonucleotides it is possible to use a single complete series of oligonucleotides or two or three replications thereof.
  • a complete series of this kind is obtained by applying chosen sequences, with a length of 40-50 nucleotides, of each one of the genes that code for the proteins (1) to (36) described above, which belong to classes (A) to (D).
  • the oligonucleotides are applied as 36 spots distributed in clearly separated individual cells on the slide.
  • the surface of the microarray must be treated before hybridization to avoid nonspecific bonds.
  • Required reagents 1. 1 XMES hybridization buffer at 42 °C. At this temperature, the solution should have a clear appearance and precipitates should disappear; if not, continue to preheat. 2. Immerse the microarray in the 1 XMES hybridization buffer for 45 minutes at 42 °C. 3. Rinse the microarray at least 5 times with sterile H 0 at ambient temperature.
  • washing buffer 1 (30 ml in a 50-ml Falcon). The cover should slide away, remove it from the Falcon. Continue the wash for 5 minutes in an oscillating agitator. 2. Wash the microarray in washing buffer 2 (0.5x SSC) for 5 minutes as above.
  • the final concentration of SDS can vary between 0.01 and 0.5% (P/V).

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

Abstract

L'invention se rapporte à une méthode de diagnostic permettant d'identifier le risque de commencement de décompensations cardiaques. Ladite méthode consiste à analyser le profil de l'expression génétique dans un échantillon biologique de façon à identifier les gènes qui sont normalement actifs dans la différentiation myocardique des cellules souches appartenant à au moins une des classes de : (A) gènes intervenant dans le développement et dans la croissance myocardique, (B) gènes déclencheurs de la mort cellulaire apoptique, et (C) gènes intervenant dans des mécanismes de survie cellulaire myocardique.
PCT/IB2002/004351 2001-10-22 2002-10-21 Methode et moyens d'identification precoce de risques de decompensation cardiaque WO2003035907A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT2001MI002205A ITMI20012205A1 (it) 2001-10-22 2001-10-22 Metodo e mezzi per l'identificazione precoce di rischi di scompenso cardiaco
ITMI2001A002205 2001-10-22

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Publication Number Publication Date
WO2003035907A2 true WO2003035907A2 (fr) 2003-05-01
WO2003035907A3 WO2003035907A3 (fr) 2004-02-12

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IT (1) ITMI20012205A1 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005114222A1 (fr) * 2004-05-13 2005-12-01 Sphingo Tec Gmbh Utilisation de precurseurs des enkephalines et/ou de leurs fragments dans les diagnostics medicaux

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6610480B1 (en) * 1997-11-10 2003-08-26 Genentech, Inc. Treatment and diagnosis of cardiac hypertrophy
US6025194A (en) * 1997-11-19 2000-02-15 Geron Corporation Nucleic acid sequence of senescence asssociated gene
JP2002541762A (ja) * 1998-11-10 2002-12-10 ボード・オヴ・リージェンツ，ザ・ユニヴァーシティ・オヴ・テキサス・システム Ｍｅｆ２転写因子の阻害による心肥大および心不全の予防方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005114222A1 (fr) * 2004-05-13 2005-12-01 Sphingo Tec Gmbh Utilisation de precurseurs des enkephalines et/ou de leurs fragments dans les diagnostics medicaux
EP2293079A3 (fr) * 2004-05-13 2011-03-30 B.R.A.H.M.S GmbH Usage des précurseurs des enképhalines et/ou leurs fragments au diagnostics médicaux
US8013123B2 (en) 2004-05-13 2011-09-06 B.R.A.H.M.S. Gmbh Use of precursors of enkephalins and/or their fragments in medical diagnostics

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WO2003035907A3 (fr) 2004-02-12
ITMI20012205A1 (it) 2003-04-22

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