EP2820131A2 - Utilisation de micro-arn ou de gènes comme marqueurs pour l'identification, le diagnostic et le traitement de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du c ur - Google Patents

Utilisation de micro-arn ou de gènes comme marqueurs pour l'identification, le diagnostic et le traitement de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du c ur

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Publication number
EP2820131A2
EP2820131A2 EP13712154.7A EP13712154A EP2820131A2 EP 2820131 A2 EP2820131 A2 EP 2820131A2 EP 13712154 A EP13712154 A EP 13712154A EP 2820131 A2 EP2820131 A2 EP 2820131A2
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Prior art keywords
seq
hsa
mir
cardiomyopathy
virus
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EP13712154.7A
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German (de)
English (en)
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Uwe Kühl
Heinz-Peter Schultheiss
Dirk Lassner
Maria ROHDE
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IKDT Institut Kardiale Diagnostik und Therapie GmbH
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IKDT Institut Kardiale Diagnostik und Therapie GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • 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
    • C12Q2600/156—Polymorphic or mutational 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • microRNAs or genes as markers for the identification, diagnosis and therapy of individual forms of non-ischemic cardiomyopathies
  • the invention relates to the use of certain microRNAs and specific genes as markers for the identification of individual forms of non-ischemic cardiomyopathies or storage diseases of the heart according to the preamble of claim 1, a diagnostic system for identifying different forms of non-ischemic cardiomyopathies or storage diseases of the heart according to the preamble of Claim 4, a method for identifying and distinguishing individual forms of non-ischemic cardiomyopathies or storage diseases of the heart according to the preamble of claim 1 1 and a drug containing microRNAs, according to the preamble of claim 13.
  • the invention further relates to novel uses of the genotype in Referring to a deletion in the CCR5 gene as a biomarker according to the preambles of claims 14 and 15.
  • Cardiovascular diseases are by far the most common cause of death in western countries today. The incidence of heart failure in Europe and the US is 12 and 15 million, respectively. With approximately 4 million (30%) patients in Europe, dilated cardiomyopathy (DCM) is the most common form of non-ischemic cardiomyopathy. The 5-year survival rate for this viral inflammatory heart disease is 50% without specific treatment. Furthermore, severe cardiac injury developed in 45% of all transplant patients on the basis of an existing DCM. The high incidence of non-ischemic cardiomyopathy and the enormous health economic consequences of these diseases require an early and specific diagnosis of the individual subforms and a targeted therapy based thereon. This applies in the same way to memory diseases of the heart, such as amyloidosis-related cardiomyopathies.
  • Cardiomyopathy refers to diseases of the heart muscle itself, which are not primarily caused as a result of other diseases of the cardiovascular system. These diseases are therefore based neither on a mechanical overload (eg due to high blood pressure or a valve defect), nor to a lack of blood flow to the coronary arteries (coronary heart disease).
  • non-ischemic cardiomyopathy and heart disease In contrast to coronary heart disease with multiple diagnostic options, there are no specific, non-invasive diagnostic parameters for the various forms of non-ischemic cardiomyopathy and heart disease. Because of their multiple origins, non-ischemic cardiomyopathy and heart disease can only be diagnosed accurately with invasive methods (myocardial biopsy) with the aim of achieving further therapy-relevant differentiation of the individual clinical pictures.
  • cardiomyopathy diagnostics The gold standard for cardiomyopathy diagnostics is myocardial biopsy, which is only performed in individual cardiology centers and in a few countries and even there only in a very limited number of patients. In addition, for the most part, only a histological examination of the heart material is carried out without the necessary immunohistochemical inflammation differentiation and the molecular-biological investigations on viral infections.
  • microRNAs have been recognized as important regulators of genetic expression, including their importance in the development of heart muscle diseases.
  • MicroRNAs are single-stranded, short ribonucleic acid molecules (RNA molecules) of 17 to 24 bases in length, which directly interfere with gene regulation. In particular, the degradation of the target RNA or its translation are controlled. MicroRNAs form a new regulatory cycle of disease-related and tissue-specific gene expression. Increasingly, circulating microRNAs in human serum or in peripheral blood cells are being used as stable biomarkers for the diagnosis of various diseases and for the monitoring of applied therapies.
  • Voellenkle et al. have described in the publication "MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients", Physiol. Genomics 42 (2010), pages 420-426, various microRNAs by which healthy individuals from patients with ischemic cardiomyopathy (ICM) or with However, a distinction between patients with ischemic cardiomyopathy and patients with non-ischemic dilated cardiomyopathy was not possible on the basis of the identified microRNAs.
  • ICM ischemic cardiomyopathy
  • ICM ischemic cardiomyopathy
  • DCM dilated cardiomyopathy
  • AS aortic stenosis
  • WO 2009/012468 A2 discloses various miRNAs as biomarkers which are used to generally diagnose heart failure.
  • the rough diagnosis described in this international patent application relates to a distinction between ischemic cardiomyopathy on the one hand and idiopathic dilated cardiomyopathy on the other hand.
  • ischemic cardiomyopathy on the one hand
  • idiopathic dilated cardiomyopathy on the other hand.
  • WO 2010/097471 proteins are described which play a role in the apoptosis of cardiomyocytes. These proteins are thereby identified via miRNAs, which are also to be used for the treatment of heart failure. However, a differential diagnosis with respect to different non-ischemic cardiomyopathies or cardiac memory diseases is not described.
  • microRNAs are used as markers for the identification of non-ischemic cardiomyopathies or memory diseases of the heart. In this way it is possible to distinguish the individual non-ischemic cardiomyopathies from one another or from storage diseases of the heart or the individual storage diseases of the heart from one another.
  • the microRNA is selected from the group consisting of the following microRNAs:
  • microRNA designations are clearly known to the person skilled in the art as unique designations of different microRNA sequences.
  • sequences standing behind these designations can be queried, for example, in the freely accessible database miRBase (accessible at the Internet address http://www.mirbase.org). They are also listed in the attached sequence listing. If individual of the abovementioned designations are used both for a stem-loop structure and for a mature microRNA sequence, the mature microRNA sequence, which is also known to the person skilled in the art under the English-language term "mature sequence", is the one behind each This is also evident from the appended sequence listing.
  • genes are used as markers to identify individual non-ischemic cardiomyopathies or memory diseases of the heart.
  • the genes are selected from the group consisting of the genes listed below, each specific portion of these genes are used, which are given in the attached sequence listing.
  • the sections used usually correspond to one or more exons of these genes.
  • the genes ATP6, CYB, ND1 and ND4 are mitochondrially encoded genes that do not have exons so that they are used in full length.
  • the TLR9 gene also uses the full-length gene.
  • the claimed invention is based on the concept of using microRNAs and / or genes as markers to identify the presence of a subtype (ie, a form) of non-ischemic cardiomyopathy or memory disease of the heart. In the present case, therefore, it is not a question of distinguishing healthy patients from sick patients or distinguishing any myocardial diseases from each other, but rather specific subgroups or forms of non-ischemic cardiomyopathies and subgroups or forms of memory diseases of the heart as well as subgroups or forms of infections of the heart Cardiac muscle, even without cardiomyopathic manifestations, to identify what clinically not possible.
  • microRNAs or genes are used in the context of a microRNA profile diagnostics or gene expression analysis, it is possible to identify patients with a non-ischemic cardiomyopathy, a memory disease of the heart or an infection of the heart muscle without cardiomyopathic manifestations in a specific subgroup and also to treat the respective clinical picture in a targeted manner at an early stage. It is possible to use the genes as complementary markers in microRNA profile diagnostics and vice versa.
  • the expression rates of the aforementioned microRNAs and genes are determined in a quantitative or semi-quantitative manner. From an increased expression of certain microRNAs and / or a reduced expression of other specific microRNAs can then diagnose a certain form of non-ischemic cardiomyopathy or a storage disease of the heart.
  • the abovementioned nucleic acids have proven to be particularly suitable for the specific diagnosis of individual non-ischemic cardiomyopathies and storage disorders of the heart. Some of the nucleic acids are so specific for certain diseases to be diagnosed that they can be used alone as specific markers. Other nucleic acids are not specific for a single disease to be diagnosed but, for example, for two diseases to be diagnosed.
  • nucleic acids are preferably not used alone, but in combination with other nucleic acids from the above list. But even with specific nucleic acids, it is advisable to refine the diagnosis of using these not individually, but in combination with other nucleic acids in the above list. In this way, the validity of diagnostic tests can be stabilized.
  • the microRNAs or genes it is also possible to use synthetic nucleic acid molecules which have an identical or complementary sequence to the abovementioned microRNAs or genes.
  • the microRNAs and / or genes of the above list are used in the form of diagnostically relevant profiles.
  • a profile may for example consist of at least or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 , 250, 300, 400, 500, 600 or all microRNAs and / or genes in the above list.
  • microRNAs are used as single markers or in diagnostic profiles of different microRNAs.
  • diagnostic profiles of different microRNAs it is also possible to use a more complex diagnostic profile of different microRNAs for the diagnosis of the various diseases to be diagnosed. This has the advantage that with a single diagnostic profile a wide variety of diagnoses can be made, such a profile is thus universally applicable within the given question.
  • the detection of the abovementioned microRNAs and the determination of the respective gene expressions can each be carried out individually.
  • Complex diagnostic systems consist of detection components for both nucleic acid groups.
  • the non-ischemic cardiomyopathies or storage disorders of the heart are selected from the group comprising virus-free dilated cardiomyopathy, virus-induced or virus-free inflammation-induced cardiomyopathy, virally-induced cardiomyopathy, cardiomyopathy with limited left ventricular ejection fraction, inflammatory cardiomyopathy, adenovirus-induced cardiomyopathy, enterovirus-induced cardiomyopathy, Coxsackievirus-induced cardiomyopathy (which is a special form of enterovirus-induced cardiomyopathy), HHV6 virus-induced cardiomyopathy, chromosomally-integrated HHV6 (ciHHV6) virus-induced cardiomyopathy, erythroid-induced cardiomyopathy, myocarditis with high incidence of inflammatory cells, giant cell myocarditis, and amyloidosis.
  • virus-free dilated cardiomyopathy in a virus-free dilated cardiomyopathy without inflammation of the myocardium or in a virus-free dilated cardiomyopathy with inflammation of the myocardium is preferably possible.
  • enterovirus (coxsackievirus) -induced cardiomyopathy in enterovirus (coxsackievirus) -induced cardiomyopathy with spontaneous clearance of the virus by the patient's own immune system or in enterovirus (coxsackievirus) -induced cardiomyopathy without spontaneous elimination of the virus to differentiate the patient's own immune system.
  • myocarditis with a high incidence of inflammatory cells can also be distinguished into acute myocarditis or borderline myocarditis. Borderline myocarditis is characterized by many inflammatory cells in the heart muscle, with no myozytenunter réelle are detectable. The aforementioned breakdowns of individual diseases into finer subclasses can be combined with each other in any manner.
  • microRNAs and genes of the above list are to be understood throughout as human sequences.
  • a restricted ejection fraction is considered to be an ejection fraction that is less than 50% of the normal ejection fraction.
  • the microRNA to be used as a marker is selected from the group consisting of all those microRNAs which score 1 or 1 for the diagnosis of a specific non-ischemic cardiomyopathy or a specific memory disease of the heart in the examinations which are explained in the examples presented below 2 has been assigned. These are the following microRNAs, which are a selection from the microRNAs listed above:
  • the nucleic acids to be used as markers are selected from the group which does not directly include those microRNAs or genes which are responsible for the diagnosis a specific non-ischemic cardiomyopathy or a memory disease of the heart in the examinations, which are explained in the examples below, the score 3 or 4 was assigned. In this case, individual, several or all of these microRNAs or genes provided with the scores 3 or 4 can be excluded from the scope of protection.
  • the invention also relates to a diagnostic system for the identification of individual non-ischemic cardiomyopathies or memory diseases of the heart.
  • a diagnostic system for the identification of individual non-ischemic cardiomyopathies or memory diseases of the heart.
  • Such a system comprises at least one probe having a sequence corresponding to or complementary to the microRNAs or genes from the group as described above.
  • the diagnostic system has at least or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, 500 or 600 such probes.
  • all the microRNAs / genes of the above list are contained in identical or complementary sequence in the diagnostic system as a probe.
  • the diagnostic system thus has a determinable number of diagnostically relevant microRNA sequences and / or gene sequences which represent a diagnostically relevant microRNA profile and / or gene profile in the system. MicroRNA sequences can be shared in any manner with gene sequences.
  • the diagnostic system has a means for detecting an additional prognostic marker.
  • this prognostic marker is a deletion in the CCR5 gene, in particular a 32 base pair deletion (CCR5 del-32 markers).
  • CCR5 del-32 markers are a positive prognostic marker for the occurrence of diabetes in patients with cardiomyopathy, a positive prognostic marker for myocardial infections with enterovirus infections and erythro virus HHV6 single and double infections (most common cardiotropic viruses) positive prognostic marker for the 7-year mortality of patients with cardiomyopathy.
  • the CCR5 del-32 marker is a positive prognostic marker (predictor) in cardiomyopathy patients for lower mortality, fewer myocardial infections, and fewer systemic diseases such as diabetes.
  • a diagnostic system for identifying the genotype related to a deletion in the CCR5 gene comprises at least one probe having a sequence which corresponds to or is complementary to a portion of the CCR5 gene.
  • the selected probe enables the determination of sequences of a gene segment within which the 32-base-pair deletion known per se occurs.
  • the diagnostic system is in the form of a kit for performing a polymerase chain reaction (PCR), wherein the probes are also provided as primers in solution.
  • PCR polymerase chain reaction
  • the probes are also provided as primers in solution.
  • carrier plates with a large number of wells (for example, 384, or even 400-3000 wells)
  • such a system allows numerous PCRs to be performed simultaneously, making this system suitable for a large number of probes.
  • the diagnostic system is in the form of a nucleic acid chip. It can be provided in particular that at least 5 probes are applied for each nucleic acid to be detected on the chip. In this way, the expression of microRNAs or genes can preferably be measured semi-quantitatively.
  • a chip has the advantage that the presence of numerous different microRNAs in the examined sample can be detected simultaneously. The number of simultaneously detectable microRNAs is still much larger than in the case of a PCR. In particular with a larger number of probes (for example more than 25, but in particular more than 100 or more than 1000 probes), the use of a chip is particularly suitable for reasons of efficiency.
  • the claimed invention also relates to a method of identifying and distinguishing individual forms of non-ischemic cardiomyopathies or cardiac memory diseases comprising the following steps. First, a sample is obtained which has been obtained from a patient suspected of having a non-ischemic cardiomyopathy or memory disease of the heart. Subsequently, the sample is contacted directly or after previous biochemical modification with at least one probe, wherein the probe has a sequence which corresponds to the sequences of the microRNAs and / or the genes from the above lists or is complementary to these. This contacting occurs under conditions that involve hybridization between in the sample Allow microRNAs and / or genes on one side and at least one probe on the other side. It is then determined whether a hybridization between the microRNAs or the genes of the sample and the at least one probe has occurred. Subsequently, it is determined on the basis of this hybridization result which microRNA (s) or genes are present in the sample.
  • the sample is preferably a total RNA extract which originates from a tissue sample of the patient which has a proportion of microRNAs in addition to the mRNA.
  • genomic DNA is used as a sample.
  • the tissue sample may comprise a whole tissue of the patient, such as myocardial tissue or blood. Alternatively, the tissue sample may also comprise only a portion of a tissue, such as red or white blood cells or blood serum or blood plasma.
  • the tissue sample may also comprise other body fluid of the patient, which need not necessarily be a tissue. The tissue sample is thus a treated or untreated sample taken from the patient. From this sample, an RNA extract is then obtained before the start of the process, which is used as a sample in the presently claimed method.
  • the invention also relates to a medicament for the treatment of non-ischemic cardiomyopathies or storage disorders of the heart, which contains as pharmaceutically active substance at least one nucleic acid having a sequence which is identical or complementary to the sequence of one of the microRNAs according to the above explanations other type affects the formation of microRNAs as explained above.
  • a medicament can serve to increase the content of a particular microRNA in the blood or in the cells of a patient so as to enhance the positive properties of this microRNA with regard to a specific clinical picture. It can also be used to eliminate a particular microRNA whose content is increased in a particular clinical picture by hybridization or a comparable interaction in order to counteract the negative effect of this microRNA on the corresponding clinical picture.
  • the selected nucleic acid or group of nucleic acids is the sole pharmaceutically active ingredient of the subject medicament.
  • the medicament is preferably suitable not only for therapeutic purposes but also for diagnostic purposes.
  • the invention also relates to the therapeutic use of the illustrated microRNAs or nucleic acid sequences which are identical or complementary to these microRNAs, in particular for the treatment or therapy of non-ischemic cardiomyopathies or storage disorders of the heart.
  • the invention further relates to the use of the genotype with respect to a deletion in the CCR5 gene as a biomarker in order to provide a decision-making aid as to whether or not beta-interferon therapy is indicated in a patient.
  • the deletion is preferably the 32-base-pair deletion known per se in the CCR5 gene.
  • the genotype is determined in vitro on the basis of a patient sample, which was previously taken from a patient; So there is no diagnosis on the body of the patient.
  • the decision-making aid offered does not constitute a therapy or a therapeutic treatment. This takes place only when, taking into account the decision-making aid offered, an independent therapy decision has been made, and is not the subject of the present invention.
  • the invention further relates to the use of the genotype with respect to a deletion in the CCR5 gene as a prognostic marker indicative of increased 7-year mortality and / or virus spontaneous clearance in cardiomyopathy patients.
  • this prognostic marker relates to patients with enterovirus (coxsackievirus) -induced cardiomyopathy.
  • Preferred embodiments of the use of the microRNAs shown above and subsequently explained in connection with the examples can also be transferred to a corresponding therapeutic application in an analogous manner. In all of this, a sequence that is at least 90%, in particular at least 95%, and most especially at least 99% identical to the sequence of one of the mentioned microRNAs or genes, considered as "identical" within the meaning of the present invention.
  • Fig. 1 is a graphical representation of the quantitatively determined differential
  • 3 is a graphical representation of the prognostic relevance of the 32 base pair deletion of the CCR5 gene to a double infection with erythroid virus and HHV6 in cardiomyopathy patients,
  • 6A is a second graph showing the prognostic relevance of the 32 base pair deletion of the CCR5 gene on 7-year mortality in cardiomyopathy patients with myocardial enterovirus infection
  • Fig. 6B is a graphic representation of the relevance of the 32 base pair deletion of
  • Fig. 6C is a graph showing the relevance of spontaneous elimination of enterovirus (coxsackievirus) on 7-year mortality in cardiomyopathy patients with myocardial enterovirus infection.
  • Fig. 6D is a graphical representation of the relevance of the 32 base pair deletion of
  • 6E is a graph showing the relevance of the number of inflammatory cells to the survival rate of myocardial cardiomyopathy patients
  • Fig. 7 is a graphical representation of the quantitatively determined differential
  • Fig. 8 is a graphical representation of the quantitatively determined differential
  • FIG. 9 is a graphical representation of the quantified differential
  • FIG. 1 1 A is a schematic overview of the possibilities of diagnosis and treatment of cardiomyopathies according to the prior art (clinical diagnostics without myocardial biopsy) and Fig. 1 1 B is a schematic overview of the possibilities of diagnosis and treatment of cardiomyopathies, taking into account the present invention.
  • Figures 1 to 5 will be explained in connection with Example 26.
  • FIGS. 6 to 9 will be explained in connection with Example 27.
  • Example 1 Diagnosis of adenovirus-induced cardiomyopathies by means of a biopsy sample
  • RNA extract an extract of the total RNA of the respective biopsy
  • microRNA components This total RNA extract was then analyzed for the presence of specific microRNAs using two variants.
  • RNA chip The RNA of the total RNA extract was labeled with biotin, applied to an RNA chip and incubated for 16 hours. Such an RNA chip is sometimes referred to as a DNA chip because it contains DNA probes. However, these are intended for RNA detection, for which reason the term "RNA chip” is used here ..
  • RNA molecules were subsequently detected with the fluorescent dye streptavidin-phycoerythrin, which binds to biotin, and by determining the fluorescence intensity of the bound streptavidin-phycoerythrin, the amount of hybridized microRNA could be determined in a semiquantitative manner.
  • RNA chips For the individual process steps, standard buffers recommended by the respective manufacturer of the RNA chips were used. These are generally familiar to the person skilled in the art. For the detection of mRNA "whole genome chips" of the company Affymetrix were used and for the microRNA among other things microRNA chips of the company febit biomed.
  • Variant 2 (PCR gene map) The microRNA contained in the total RNA extract was used for the synthesis of cDNA. Depending on the amount of cDNA obtained, this was pre-amplified if necessary. Subsequently, the cDNA was applied to a card and a quantitative PCR was performed. In this way, a relative quantification of the individual cDNAs in real time on the relative fluorescence to mitampl en budget genes (ie to constitutively expressed microRNAs) take place.
  • microRNAs were identified whose expression was modified compared to samples derived from patients with other non-ischemic cardiomyopathies or memory disease of the heart.
  • Table 1 below lists the identified microRNAs whose expression is modified in adenovirus-induced cardiomyopathy. Furthermore, this table indicates the nature of this modification. For this purpose, a point value was calculated taking into account the results explained in the following examples and it was determined whether the expression of the respective microRNA was high or downregulated.
  • a score of 1 means that the observed expression modification is specific for one of the considered non-ischemic cardiomyopathies or memory diseases of the heart and only occurs significantly changed in this.
  • MicroRNAs with a score of 1 are therefore specific markers for adenovirus-induced cardiomyopathy. Their marker function can exert these microRNAs by an increased expression (upregulation) or a reduced expression (downregulation).
  • a score of 1 indicates microRNAs that allow for unambiguous identification of a disease, even if it is not known what the observed modification of expression is.
  • a score of 2 means that the observed expression modification is specific for two of the non-ischemic cardiomyopathies or memory diseases of the heart considered and occurs only in these two diseases. The expression is increased in the case of the first of these two diseases and lowered in the case of the second of these two diseases.
  • MicroRNAs that have a score of 2 are therefore specific markers for adenovirus-induced cardiomyopathy, if it is additionally known whether their expression is up-regulated or downregulated.
  • a score of 2 denotes microRNAs which allow a clear identification of a disease, if it is additionally known which species is the observed modification of the expression.
  • a score of 3 means that the observed expression modification is significant for several of the non-ischemic cardiomyopathies or memory diseases of the heart considered.
  • the expression in the case of two diseases is the same modified, ie either increased or decreased.
  • the expression is inversely modified, either decreased or increased.
  • MicroRNAs which are assigned a score of 3, are therefore in themselves nonspecific markers for adenovirus-induced cardiomyopathy, since they could also indicate a different clinical picture.
  • another microRNA marker which is either specific or also non-specific, a clear determination of an adenovirus-induced cardiomyopathy can already be made possible.
  • a score of 4 means that the observed expression modification is significant for exactly two of the non-ischemic cardiomyopathies or memory diseases of the heart considered.
  • the expression is in both cases the same way modified, so either increased or decreased.
  • MicroRNAs which are assigned a score of 4 are therefore in themselves nonspecific markers for adenovirus-induced cardiomyopathy, since they could also indicate a different clinical picture.
  • another microRNA marker which is either specific or also non-specific, a clear determination of an adenovirus-induced cardiomyopathy can already be made possible.
  • this applies only if its expression is modified in another disease other than that of the further disease in which the expression of the first non-specific marker is likewise modified.
  • the invention relates to the use of the microRNAs provided in Table 1 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of adenovirus-induced cardiomyopathy.
  • Example 2 Diagnosis of enterovirus-induced (coxsackievirus-induced)
  • Cardiomyopathies by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from enterovirus-induced cardiomyopathy or coxsackievirus-induced cardiomyopathy as a special case of enterovirus-induced cardiomyopathy.
  • the identified microRNAs whose expression is modified in such enterovirus (coxsackievirus) induced cardiomyopathy are listed in Table 2 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • Example 3 Diagnosis of enterovirus (coxsackievirus) -induced cardiomyopathies with spontaneous elimination of the virus by the patient's immune system using a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from enterovirus (coxsackievirus) -induced cardiomyopathy, but later a spontaneous elimination of the virus by the patient's own immune system took place. These patients do not require drug treatment of the viral infection.
  • enterovirus coxsackievirus
  • Table 3 The identified microRNAs whose expression is modified in such enterovirus (coxsackievirus) induced cardiomyopathy are listed in Table 3 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of microRNAs provided with a score of 1 and / or 2 in Tables 2 or 3 individually or in any combination with each other as markers for the identification of enterovirus (coxsackievirus) -induced cardiomyopathy, especially one Enterovirus (coxsackievirus) -induced cardiomyopathy with spontaneous elimination of the virus by the patient's own immune system.
  • enterovirus coxsackievirus
  • Example 4 Diagnosis of enterovirus (coxsackievirus) -induced cardiomyopathies without spontaneous elimination of the virus by the patient's immune system by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from enterovirus (coxsackievirus) -induced cardiomyopathy, but in which later no spontaneous elimination of the virus by the patient's own immune system took place, so that a virus persistence in need of treatment developed.
  • enterovirus coxsackievirus
  • the invention relates to the use of the microRNAs provided with a score of 1 and / or 2 in Table 4 individually or in any combination with one another as markers for the identification of enterovirus (coxsackievirus) -induced cardiomyopathy, in particular an enterovirus (Coxsackievirus-) induced cardiomyopathy without spontaneous elimination of the virus by the patient's own immune system.
  • Example 5 Diagnosis of amyloidosis of the heart by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy samples were taken from patients suffering from amyloidosis of the heart.
  • Amyloidosis is a typical memory disorder of the heart and is characterized by amyloid deposits in the heart muscle.
  • the invention relates to the use of the microRNAs provided in Table 5 with a score of 1 and / or 2 individually or in any combination with each other as markers for identifying a storage disease of the heart, in particular an amyloidosis of the heart.
  • Example 6 Diagnosis of myocarditis with high incidence of inflammatory cells by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy samples were taken from patients who were suffering from an intramyocardial inflammation with a high incidence of inflammatory cells. An increased influx of inflammatory cells is present when there are more than 14 inflammatory cells per mm 2 of tissue in the inflamed tissue. This is called borderline myocarditis. If it is also possible to detect histopathologically myocyte deaths, this is called active / acute myocarditis. Active myocarditis is also present if histological evidence of an inflammatory cell-associated loss of cardiomyocytes is present without exceeding the above-mentioned cell number.
  • the invention relates to the use of the microRNAs provided in Table 6 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a myocarditis with a high incidence of inflammatory cells.
  • Example 7 Diagnosis of giant cell myocarditis by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy samples were taken from patients suffering from giant cell myocarditis.
  • a giant cell myocarditis is characterized by the appearance of polynuclear giant cells accompanying acute myocarditis in the heart muscle. Since untreated giant cell myocarditis usually takes a fatal course, this form of acute myocarditis must be accurately determined, which is currently possible only by histological review of paraffin sections. However, the detection of multinucleated giant cells rarely succeeds. In suspected cases often 10 or more heart muscle biopsies must be cut open so that a histological evidence can be found, which almost no cardiology center performs worldwide. The identified microRNAs whose expression is modified in such giant cell myocarditis are listed in Table 7 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of the microRNAs provided in Table 7 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a giant cell myocarditis.
  • Example 8 Diagnosis of a virus-free, dilated cardiomyopathy with or without inflammation of the myocardium by means of a biopsy sample
  • the procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from a virus-free, dilated cardiomyopathy with inflammation of the myocardium or from a virus-free dilated cardiomyopathy without inflammation of the myocardium.
  • the invention relates to the use of the microRNAs provided in Table 8 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a virus-free dilated cardiomyopathy.
  • Example 2 The procedure was analogous to Example 1, except that the biopsy samples were taken from patients suffering from erythroid-induced cardiomyopathy.
  • the erythroid virus causing such cardiomyopathy occurs in two subtypes, genotype 1 being known as parvovirus B19 and erythroid virus B19, respectively.
  • genotype 1 being known as parvovirus B19 and erythroid virus B19, respectively.
  • the parvovirus plays only a minor role, so that in the present case no distinction should be made of the erythroid virus in its genotypes.
  • the identified microRNAs whose expression is modified in such erythroid-induced cardiomyopathies are listed in Table 9 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of the microRNAs provided in Table 9 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of an erythroid-induced cardiomyopathy.
  • Example 2 The procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from erythroid-induced cardiomyopathy without inflammation of the myocardium and with a normal ejection fraction.
  • the invention relates to the use of the microRNAs provided in Table 10 with a score of 1 and / or 2 individually or in any combination with one another as markers for the identification of an erythroid-induced cardiomyopathy, in particular an erythroid-induced cardiomyopathy without inflammation of the myocardium and with normal ejection fraction.
  • Example 1 Diagnosis of erythroid-induced cardiomyopathy with inflammation of the myocardium and with restricted ejection fraction by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from an erythroid-induced cardiomyopathy with inflammation of the myocardium and with a restricted ejection fraction.
  • the ejection fraction observed in the patients was less than 50% of a normal ejection fraction.
  • the identified microRNAs whose expression is modified in such erythroid-induced cardiomyopathies are listed in Table 1 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of the microRNAs provided in Table 11 with a score of 1 and / or 2 individually or in any combination with one another as markers for the identification of an erythroid-induced cardiomyopathy, in particular of an erythroid-induced cardiomyopathy Inflammation of the myocardium and with limited ejection fraction.
  • Example 12 Diagnosis of inflammation-induced cardiomyopathy by means of a
  • biopsy sample The procedure was analogous to Example 1, except that the biopsy specimens were taken from patients suffering from inflammation-induced cardiomyopathy.
  • the invention relates to the use of the microRNAs provided in Table 12 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of an inflammation-induced cardiomyopathy.
  • Example 13 Diagnosis of Virally Induced Cardiomyopathy Using a Biopsy Sample The procedure was analogous to Example 1, except that the biopsy samples were taken from patients suffering from a viral-induced cardiomyopathy (caused by an erythroid virus or an enterovirus). The identified microRNAs whose expression is modified in such virally induced cardiomyopathies are listed in Table 13 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of the microRNAs provided in Table 13 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a virally induced cardiomyopathy.
  • Example 14 Diagnosis of myocarditis with high incidence of inflammatory cells by means of a blood cell sample
  • the detection of specific gene expression or microRNAs for various forms of non-ischemic cardiomyopathy can also be done in blood cells.
  • the corresponding pattern of expressed nucleic acids in blood cells differs significantly from that in cardiac muscle biopsies.
  • the invention relates to the use of the microRNAs provided in Table 14 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a myocarditis with a high incidence of inflammatory cells.
  • Example 15 Diagnosis of myocarditis with high incidence of inflammatory cells without myeloid (borderline myvocarditis) by means of a blood cell sample
  • Example 14 The procedure was analogous to Example 14, except that the blood cell samples were taken from patients who had increased inflammatory cells without histological detection of myocyte sinking (borderline myocarditis) in the myocardium.
  • the identified microRNAs whose expression is modified in such myocarditis with a high influx of inflammatory cells are listed in Table 15 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • Example 16 Diagnosis of myocarditis with high incidence of inflammatory cells with detectable Mvozvtenunterqänqen (active / acute myocarditis) using a blood cell sample
  • Example 14 The procedure was analogous to Example 14, except that the blood cell samples were taken from patients who had an increased number of inflammatory cells with a histological detection of Myozytenunter réelle in heart muscle.
  • Example 17 Diagnosis of an inflammatory heart muscle by means of a blood cell sample
  • Example 14 The procedure was analogous to Example 14, except that the blood cell samples were taken from patients who had no increased incidence of inflammatory cells in the heart.
  • microRNAs whose expression is present in an inflammation-free myocardium are listed in Table 17 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of the microRNAs provided in Table 17 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of an inflammation-free myocardium.
  • Example 18 Diagnosis of cardiac amyloidosis by means of a blood cell sample
  • the procedure was analogous to Example 14, except that the blood cell samples were taken from patients who were suffering from amyloidosis of the heart.
  • the invention relates to the use of the microRNAs provided in Table 18 with a score of 1 and / or 2 individually or in any combination with one another as markers for identifying a memory disorder of the heart, in particular an amyloidosis of the heart.
  • Example 19 Diagnosis of a virus-free dilated cardiomyopathy with or without inflammation of the myocardium by means of a blood cell sample
  • the procedure was analogous to Example 14, except that the blood cell samples were taken from patients who were suffering from a virus-free dilated cardiomyopathy with inflammation of the myocardium or a virus-free dilated cardiomyopathy without inflammation of the myocardium.
  • the invention relates to the use of the microRNAs provided in Table 19 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a virus-free dilated cardiomyopathy.
  • Example 20 Diagnosis of a ciHHV6-Induced Cardiomyopathy by means of a
  • ciHHV6 denotes a special form of human herpesvirus 6. This virus has the property of being incorporated into the chromosomes of the host; This is called chromosomal integration of HHV6 (ciHHV6).
  • the invention relates to the use of the microRNAs provided in Table 20 with a score of 1 and / or 2 individually or in any combination with one another as markers for identifying an HHV6-induced cardiomyopathy, in particular a cardiomyopathy with ciHHV6 expression.
  • Example 21 Diagnosis of a virus-free dilated cardiomyopathy without inflammation of the myocardium by means of a blood serum sample
  • the detection of specific microRNAs for various forms of non-ischemic cardiomyopathy can also be done in serum.
  • the corresponding pattern of expressed nucleic acids in serum differs from that in cardiac muscle biopsies or in blood cells.
  • RNA extract was then obtained analogously to Example 1 and further processed and analyzed according to the methods described there.
  • Table 21 Expression modifications of microRNAs in dilated cardiomyopathy with or without inflammation of the myocardium.
  • the invention relates to the use of the microRNAs provided in Table 21 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a virus-free dilated cardiomyopathy.
  • Example 22 Diagnosis of myocarditis with high incidence of inflammatory cells by means of a blood serum sample
  • Example 21 The procedure was analogous to Example 21, except that the blood serum samples were taken from patients suffering from myocarditis with a high incidence of inflammatory cells. Analogously, this includes the histological detection of inflammation-cell-associated death of cardiomyocytes without exceeding the above-mentioned number of cells.
  • the invention relates to the use of the microRNAs provided in Table 22 with a score of and / or 2 individually or in any combination with one another as a marker for the identification of myocarditis with a high incidence of inflammatory cells.
  • Example 23 Diagnosis of myocarditis with high incidence of inflammatory cells without myocyte subset by means of a blood serum sample The procedure was analogous to Example 21, except that the blood serum samples were taken from patients suffering from myocarditis with a high incidence of inflammatory cells.
  • the identified microRNAs whose expression is modified in such myocarditis with a high incidence of inflammatory cells are listed in Table 23 below. With regard to the scores, reference is made to the Explanatory Notes to Example 1.
  • the invention relates to the use of the microRNAs provided in Table 23 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of a myocarditis with a high incidence of inflammatory cells, but without myocyte declines.
  • Example 24 Diagnosis of myocarditis with high incidence of inflammatory cells and cell subsets in the heart muscle by means of a blood serum sample
  • the procedure was analogous to Example 21, except that the blood serum samples were taken from patients who had a myocarditis with histological evidence of inflammatory cell-associated death of cardiomyocytes, even without exceeding the above-mentioned cell number.
  • the invention relates to the use of microRNAs provided in Table 24 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of myocarditis with a high incidence of inflammatory cells and simultaneous cell deaths in the myocardium.
  • EXAMPLE 25 Diagnosis of an Inflammatory Heart Muscle Using a Blood Serum Sample The procedure was analogous to Example 21, except that the blood serum samples were taken from patients who had no increased influx of inflammatory cells of the heart.
  • the invention relates to the use of microRNAs provided in Table 25 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of myocarditis without increased influx of inflammatory cells.
  • Example 26 CCR5 del 32 as an independent prognostic marker in patients with cardiomyopathies
  • cardiomyopathies are also developed by viral infections of the heart or inflammation-associated processes in the affected myocardium.
  • a known polymorphism in the CC chemokine receptor 5 (CCR5) gene which involves a deletion of 32 base pairs in this gene, results in a functionally impaired macrophage coreceptor. This provides protection against viral infections. Furthermore, the inflammatory response in systemic diseases is thereby modulated.
  • This 32 base pair deletion of the CCR5 gene is described, for example, by Prahalad: "Negative association between the chemokine receptor CCR5-A32 polymorphism and rheumatoid arthritis: A meta-analysis", Genes Immun. 2006, 7 (3): 264-268 and the literature cited therein.
  • FIG. 1 shows a graphic representation of the quantitatively determined differential gene expression of various genes in cardiomyopathy patients. The names of the examined genes are indicated on the x-axis. Gene expression of the genes under consideration in patients whose CCR5 gene was wild-type (no deletion was present in both alleles of the gene) was set to 100%.
  • FIG. 2 shows a graphic representation of the prognostic relevance of the 32 base pair deletion of the CCR5 gene to diabetes in cardiomyopathy patients. Of 427 patients examined, 46 were diabetics. 41 of these diabetics showed the wild type in the CCR gene.
  • FIG. 3 shows a graphical representation of the prognostic relevance of the 32 base pair deletion of the CCR5 gene to a double infection with erythroid virus and HHV6 (the two most frequently detected myocardium viruses) in patients with clinically manifest dilated cardiomyopathy (DCM).
  • DCM clinically manifest dilated cardiomyopathy
  • FIG. 4 shows a graphic representation of the prognostic relevance of the 32 base pair deletion of the CCR5 gene to enterovirus infection in cardiomyopathy patients.
  • DCM dilated cardiomyopathy
  • Figure 5 shows a graphical representation of the prognostic relevance of the 32 base pair deletion of the CCR5 gene on the 7-year mortality in cardiomyopathy patients. Mortality data were available in 268 patients. 195 wild-type patients also lived within 7 years, while 19 wild-type patients died during this period. In a total of 50 patients heterozygous for the 32 base pair deletion of the CCR5 gene and a total of 4 patients homozygous for the 32 base pair deletion of the CCR5 gene, no deaths occurred within 7 years.
  • Example 26A CCR5 del 32 as a prognostic marker in patients with enterovirus-induced cardiomyopathy The procedure was analogous to Examples 3, 4 and 26. That is, biopsy specimens suffering from enterovirus (coxsackievirus) -induced cardiomyopathy were taken from such patients. On the one hand, those patients were selected in whom a spontaneous elimination of the virus by the patient's own immune system took place later.
  • FIG. 6A shows a graphical representation of the prognostic relevance of the 32 base pair deletion of the CCR5 gene on the 7-year mortality in cardiomyopathy patients. As can be seen from these data, no patient with the 32 base pair deletion of the CCR5 gene dies. Genes (genotype hetero or mutation) in the 7-year period. In contrast, approximately 24% of patients with wild-type genotypes die.
  • Figure 6B shows a graphic representation of the relevance of the 32 base pair deletion of the CCR5 gene on a spontaneous elimination of Enterovirus (coxsackievirus) in cardiomyopathy patients with myocardial enterovirus infection.
  • Enterovirus coxsackievirus
  • FIG. 6C shows a graphic representation of the relevance of spontaneous elimination of enterovirus (coxsackievirus) on 7-year mortality in cardiomyopathy patients myocardial enterovirus infection.
  • enterovirus coxsackievirus
  • CCR5 genotype in a proven myocardial enterovirus infection allows the decision on the necessity of interferon therapy and thus a prognosis for mortality in untreated patients in the sense of personalized medicine or as a companion diagnostic for the pharmaceutical beta.
  • Interferon Example 26B CCR5 del 32 as Prognostic Marker in Patients with Respiratory Virus (Parvorius B19) Induced Cardiomyopathy
  • the procedure was analogous to Examples 3, 4 and 26. That is, biopsy specimens suffering from erythroid virus (Parvorius B19) -induced cardiomyopathy were taken from such patients. Furthermore, the 5-year mortality was determined for these patients. In these patients, the CCR5 genotype (wild type - without altering the gene sequence, hetero or mutation - a 32 base pair deletion in this gene on at least one chromosome) was determined.
  • Figure 6D shows a graphic representation of the relevance of the 32 base pair deletion of the CCR5 gene on the survival of cardiomyopathy patients with myocardial erythroid infection.
  • FIG. 6E shows a graphic representation of the relevance of the number of inflammatory cells on the survival rate of cardiomyopathy patients with myocardial erythroid infection.
  • Patients with wild-type genotype and inflammatory infiltrates less than or equal to 10 cells per mm 2 of myocardial surface die significantly less frequently than patients with the same CCR5 genotype but high numbers of inflammatory cells in the myocardium.
  • the significance of myocardial erythroid virus infection is based on a frequency of 50% virus-positive myocardial biopsies. On average, up to 90% of all patients have the wild-type CCR5 genotype. Of 4 million DCM patients in Europe, approximately 1 .8 million have the wild-type CCR5 genotype.
  • the "CCR5 genotype” biomarker is a prognostic marker in predictive myocardial erythroid infection to predict the survival of cardiomyopathy patients, and the number of inflammatory cells can be used as a prognostic marker to predict the survival of cardiomyopathy patients
  • the present invention relates to the use of these prognostic markers in order to predict the survival rate of corresponding patients.
  • Example 27 Differential regulation of genes in biopsy specimens in active myocarditis, giant cell myocarditis and qranulomatous giant cell myocarditis
  • a giant cell myocarditis is characterized by the appearance of polynuclear giant cells accompanying acute myocarditis in the heart muscle. Since untreated giant cell myocarditis usually takes a fatal course, this form of acute myocarditis must be accurately determined, which is currently possible only by histological review of paraffin sections. However, the detection of multinucleated giant cells rarely succeeds. In suspected cases, 10 and more heart muscle biopsies would have to be cut open in order to find a histological record, which is almost impossible to do anywhere in the cardiology center.
  • FIG. 7 shows a graphic representation of the differential gene expression of various genes in cardiomyopathy patients which has been quantitatively determined during an initial examination.
  • TLR genes are downregulated compared to the control group Vneg.
  • some genes can be seen, whose expression is increased in the granulomatous giant cell myocarditis, in the giant cell myocarditis without granuloma but not. These genes are also useful for distinguishing between the two diseases.
  • the threshold from which an increase in gene expression is to be taken into account can be largely determined as far as statistically significant results can still be obtained by applying this threshold.
  • FIG. 8 shows a graphic representation of the differential gene expression of various genes in cardiomyopathy patients which is quantitatively determined during a follow-up examination.
  • the follow-up examination took place between 1 month and 2 years after the initial examination.
  • the abbreviations used correspond to the designations used in FIG.
  • the threshold should preferably be set at less than 800% of the usual gene expression.
  • the genes CCL20, IL17D, IL23R, TLR8, TNF, ND4, CPT1 and CYB are suitable for a corresponding distinction.
  • FIG. 9 shows a graphic representation of the quantitatively determined differential gene expression of various genes in cardiomyopathy patients with giant cells with granuloma in a first examination and a follow-up examination.
  • the follow-up examination took place between 1 month and 2 years after the initial examination.
  • the procedure was analogous to the procedure explained with reference to FIG. It showed that, among other things, the genes CCR5, F0XP3, IFNB1, IL23R, TLR5, TLR7, ND4, ATP6, CYB and ND1 were significantly more or less expressed in the follow-up examination than was the case at the initial examination of the patients. Therefore, these genes are also suitable individually or in combination with each other for the diagnosis and monitoring of giant cell myocarditis with granuloma.
  • FIG. 10 shows a further graphical representation of the quantitatively determined differential gene expression of various genes in cardiomyopathy patients with giant cells without granuloma in a first examination and a follow-up examination.
  • the follow-up examination took place between 1 month and 2 years after the initial examination.
  • the procedure was analogous to the procedure explained with reference to FIG.
  • genes CCL20, CCR5, CCR6, FOXP3, IFNB1, IL10, IL17D, IL23R, IL6, IL6R, TGFB1, TLR3, TLR7, TLR8, TNF, ND4, ATP6, CPT1, TLR9, IL1 B, ND1 and UQCR were significantly more or less expressed in the follow-up study than was the case with the initial examination of the patients. Therefore, these genes are also suitable individually or in combination with each other for the diagnosis and monitoring of giant cell myocarditis without granuloma.
  • Tables 26 and 27 below show a list of the quantitatively determined differential gene expression of various genes in cardiomyopathy patients (analogous to the results shown in FIGS. 7, 8 and 9).
  • Patients with acute / active myocarditis (MCA) patients with giant cell myocarditis (RZ) without granuloma and patients with giant cell myocarditis with granuloma (granulomatous giant cell myocarditis) were studied.
  • the list refers to genes that can be used for differential primary diagnosis of myocarditis (Table 26) and for subsequent follow-up diagnoses for monitoring acute / active myocarditis in giant cell myocarditis with and without granuloma (Table 27).
  • the examination was carried out on RNA obtained from myocardial biopsies. In one variant, only those genes are used individually or in any combination as a marker to which a score of 1 and / or 2 was assigned in Table 26 and / or Table 27.
  • Example 28 Diagnosis of veterinary virus-induced cardiomyopathies by means of a biopsy sample
  • Example 2 The procedure was analogous to Example 1, except that the biopsy samples were taken from patients suffering from virus replication of erythroid-induced cardiomyopathy. Virus replication can be detected by virus RNA in patient tissue.
  • the invention relates to the use of the microRNAs provided in Table 28 with a score of 1 and / or 2 individually or in any combination with each other as markers for the identification of an erythroid virus-induced cardiomyopathy with virus replication.
  • FIG. 11A shows a schematic overview of the possibilities currently existing in the prior art for the diagnosis and treatment of the various forms of non-ischemic cardiomyopathies.
  • DCM dilated cardiomyopathy
  • FIG. 11b shows a schematic overview of the possibilities which exist for the diagnosis and treatment of different cardiomyopathies when the present invention is taken into account in the diagnosis. Because then it is no longer non-specifically differentiated into "healthy heart” and "sick heart", rather there is a fine diagnosis with regard to the different types of cardiomyopathy. This will be explained in more detail below with reference to FIG. 11B.
  • a cardiomyopathy that occurs in a patient can have different causes. For example, a virus infection, an inflammation or a storage disorder or an autoimmune disease can be the cause. By an initial diagnosis of cardiomyopathy as to whether there is inflammation or no inflammation, an initial differential diagnosis can already be made with regard to the cardiomyopathy that has occurred in the patient. Cardiomyopathies in which there are no signs of inflammation can be virus-associated or virus-free. In the case of virus-negative cardiomyopathies one can distinguish between cardiomyopathies with a low myocardial damage and those with a severe myocardial damage.
  • a virus-negative cardiomyopathy with a low myocardial damage can also be termed a healed myocarditis, which requires no further treatment.
  • the patient can be protected from the use of a nonspecific conventional heart failure therapy by appropriate diagnosis.
  • a virus-negative cardiomyopathy without inflammation with a pronounced myocardial damage is a dilated cardiomyopathy, which can then be treated by conventional heart failure therapy.
  • a patient has been diagnosed with cardiomyopathy in which no inflammation has been detected, but the presence of viruses has been diagnosed, this indicates a viral myocardial disease which, in addition to conventional heart failure therapy, may or may not be treated with antiviral therapy.
  • a virus-positive and a virus-negative course In a virus-positive course there is again a chronic viral heart muscle disease, so that in turn offers an antiviral therapy in addition to a conventional heart failure therapy.
  • a virus-negative course of an inflammatory Cardiomyopathy can be differentiated into cardiomyopathy with severe myocardial damage and cardiomyopathy with low myocardial damage.
  • Such cardiomyopathy with a severe myocardial damage is an inflammatory cardiomyopathy in which in addition to a conventional heart failure therapy and an immunosuppressive therapy should be performed.
  • a myocarditis which differs in the phenotype of a dilated cardiomyopathy of unclear genesis. Likewise, however, it will be treated by conventional heart failure therapy. Additional immunosuppressive therapy is also possible.
  • FIGS. 11A and 11B it is clear what advantages a fine diagnosis of different cardiomyopathies or storage disorders of the heart entails. Because only by means of such a fine diagnosis, it is possible to offer each patient a targeted therapy that offers the best possible chance of recovery. In addition, an appropriate and ineffective therapy of certain cardiomyopathies can be avoided by means of an appropriate fine diagnosis, which both relieves the patient's body and also ensures lower expenditure in the health system.
  • Table 29 below shows an overview of the different cardiomyopathies which can be distinguished from one another in the context of the present invention.
  • most of these diseases can also be diagnosed by biopsy diagnostics using classical histology, possibly supplemented by a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • this is far more complicated than a miRNA diagnosis.
  • no coxsackievirus-induced cardiomyopathy with spontaneous elimination and no coxsackievirus-induced cardiomyopathy with virus persistence can be diagnosed by the classical methods. This is only possible by means of the miRNA diagnosis presented here.
  • the Therapy Option column identifies various options for treating the particular cardiomyopathy, with the treatment options listed in non-highlighted fields being real options, ie therapies that can be made, whereas the therapy options shaded as "indications" represent such therapies that need to be done so as not to endanger the patient's life.
  • the therapy options shaded as "indications” represent such therapies that need to be done so as not to endanger the patient's life.

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Abstract

L'invention concerne l'utilisation de certains micro-ARN et/ou gènes, individuellement ou en association sous forme de profils comme marqueurs pour l'identification de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du cœur, ainsi que leur utilisation dans des systèmes de diagnostic pour l'identification de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du cœur, ces systèmes présentant au moins une partie de ces micro-ARN et/ou gènes. L'invention concerne en outre un procédé d'identification et de différenciation de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du cœur au moyen d'au moins certains de ces micro-ARN et/ou gènes. L'invention concerne par ailleurs un médicament qui contient au moins un acide nucléique présentant une séquence identique à la séquence d'un de ces micro-ARN ou complémentaire de celle-ci.
EP13712154.7A 2012-02-27 2013-02-26 Utilisation de micro-arn ou de gènes comme marqueurs pour l'identification, le diagnostic et le traitement de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du c ur Ceased EP2820131A2 (fr)

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PCT/EP2013/053800 WO2013127782A2 (fr) 2012-02-27 2013-02-26 Utilisation de micro-arn ou de gènes comme marqueurs pour l'identification, le diagnostic et le traitement de formes individuelles de cardiomyopathies non ischémiques ou de maladies de surcharge du cœur

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