HK1251019B - Signature of health - Google Patents

Description

Health Sign

Technical Field of the Invention

The present invention relates to a method, use and kit for diagnosing changes in a subject's health condition based on the measurement of miRNA expression profiles.

Background of the Invention

Biomarkers now play a key role in the diagnosis, risk stratification and treatment management of various diseases. MicroRNA (miRNA) is a new type of biomarker. They represent a group of small non-coding RNAs that regulate gene expression at the post-translational level by degrading or blocking the translation of messenger RNA (mRNA) targets. It has been found that miRNA is expressed in a highly tissue-specific manner, and miRNA is also present in body fluid samples, including blood. However, it is not clear that miRNA is present in the mechanism of blood, particularly blood cells, or its function in these blood fractions (fractions). Particularly desirable are non-invasive biomarkers that allow rapid, convenient and cost-effective diagnosis/prognosis, thereby avoiding the need for surgical intervention. In particular, miRNA has not yet been systematically evaluated as a non-invasive biomarker for the potential role of changes in the health status of a general diagnosis object. Therefore, there is still a need for an effective method and a test kit for changes in the health status of a non-invasive diagnosis object.

The present inventors have for the first time evaluated miRNA expression at the genome-wide level in a broad range of healthy subjects (n=158) or subjects diagnosed with various diseases (n=883), including cancer (i.e., lung cancer, colon cancer, kidney cancer, glioblastoma, prostate cancer, melanoma), neurodegenerative diseases (i.e., multiple sclerosis, Parkinson's disease), cardiovascular diseases (acute myocardial infarction, heart failure), autoimmune diseases (i.e., sarcoidosis, psoriasis), or inflammatory diseases (i.e., COPD, BPH). They surprisingly found that in blood samples, preferably whole blood samples or blood cell fractions containing red blood cells, white blood cells, and/or platelets, certain miRNAs (disease-regulated miRNAs, FIG1 ) were significantly deregulated between healthy controls and diseased subjects, whereas certain other miRNAs (disease-maintaining miRNAs, FIG2 ) were not significantly deregulated between healthy controls and diseased subjects. Therefore, the combination of the disease-regulated and disease-maintaining miRNAs is suitable non-invasive biomarkers for diagnosing changes in a subject's health status. In particular, disease-maintaining miRNAs can be used as internal normalizers for disease-modulated miRNAs, which is very advantageous when aiming to bring reliable and robust diagnostic tests to the market, since such tests cannot rely on overall normalization methods due to the use of a set of only a few miRNA biomarkers. Therefore, the test according to the present invention can rely on comparing relative expression levels with a reference. In addition, the inventors of the present invention explored the use of miRNAs from blood cells of various blood cell fractions comprising red blood cells, white blood cells and/or platelets (which were obtained from collected whole blood samples). Therefore, the miRNAs of all blood cells representing the components of the peripheral immune system were analyzed. Therefore, the diagnostic content evaluated by this method refers to the evaluation of miRNA-biomarker information of the peripheral immune system, which is different from the methods based on miRNAs from extracellular blood fractions (serum, plasma), which are directly related to the diseased organs or tissues. Furthermore, methods that rely on miRNA from extracellular blood fractions (serum, plasma) are highly prone to contamination by miRNA leaking from blood cells into the extracellular matrix during collection and/or pre-analytical processing, which is not the case when using miRNA biomarkers derived from blood cells or blood cell fractions.

Therefore, the disease-modulating miRNAs in combination with the disease-maintaining miRNAs from blood cells or blood cell fractions comprising red blood cells, white blood cells and/or platelets are analytically robust and reliable appropriate non-invasive biomarkers that have internal normalization capabilities for changes in the health status of the diagnosed subject using orthogonal diagnostic information derived from the peripheral immune system.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method for diagnosing a health condition of a subject, comprising the steps of:

(a) determining the expression profile of a first predetermined group of one or more miRNAs in a blood sample from a subject, wherein the miRNAs included in the first predetermined group of miRNAs are selected from the miRNAs listed in Figure 1 and/or Figure 3,

(b) determining the expression profile of a second predetermined group of one or more miRNAs in the blood sample from the subject, wherein the miRNAs included in the second predetermined group of miRNAs are selected from the miRNAs listed in Figure 2 and/or Figure 4,

(c) normalizing the expression profile of the first predetermined group of miRNAs to that of the second predetermined group of miRNAs,

(d) comparing the normalized expression profile from step (c) with a reference,

(e) diagnosing a change in the subject's health condition when the comparison with the reference changes.

In a second aspect, the present invention provides a method for diagnosing a health condition of a subject, comprising the steps of:

(a) determining the expression profile of a first predetermined set of miRNAs in a blood sample from a subject,

(b) determining the expression profile of a second predetermined set of miRNAs in said blood sample from said subject,

(c) comparing the expression profile of step (a) and the expression profile of step (b) with a reference expression profile,

(d) diagnosing a change in the subject's health condition when the comparison to the first reference expression profile is changed and the comparison to the second reference expression profile is unchanged.

In a third aspect, the present invention provides the method according to the first aspect and the second aspect of the present invention for the following uses:

(i) comparing the health status of one or more subjects,

(ii) monitoring the health status of the subject,

(iii) monitoring the status of the subject's immune system,

(iv) monitoring the subject's response to therapeutic treatment, preferably pharmaceutical treatment.

In another aspect of the present invention, the inventors found that in blood samples, preferably in whole blood samples or blood cell fractions containing red blood cells, white blood cells and/or platelets, certain miRNAs (cancer-regulated miRNAs, Figure 3) were significantly deregulated between healthy controls and cancer subjects, while certain other miRNAs (cancer-maintained miRNAs, Figure 4) were not significantly deregulated between healthy controls and cancer subjects.

In other aspects, the present invention provides a method for monitoring the health of a subject, comprising the steps of:

(a) diagnosing the health status of a subject at a first time point by implementing the method according to the first aspect or the second aspect,

(b) diagnosing the health status of the subject at one or more later time points by performing the method according to the first aspect or the second aspect, and

(c) comparing the health condition diagnosed in step (a) with the health condition diagnosed in step (b), thereby monitoring the health condition of the subject.

This summary of the invention does not necessarily describe all features of the invention. Other embodiments will become apparent from a review of the detailed description that follows.

Detailed Description of the Invention

definition

Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

Hereinafter, key elements of the present invention will be described. These key elements are listed together with specific embodiments, however, it should be understood that they can be combined in any way and in any number to produce other embodiments. The embodiments and preferred embodiments of the different descriptions should not be construed as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass such embodiments, which combine the disclosed and/or preferred key elements of the embodiments clearly described with any number. In addition, any arrangement and combination of all described key elements in this application should be considered to be disclosed by the application's specification sheet, unless context otherwise indicates.

Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

To practice the present invention, conventional chemical, biochemical and recombinant DNA techniques are employed, unless otherwise indicated, which are described in the literature in the field (see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate the disclosure herein by prior invention.

Throughout the specification and appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a recited integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "test compound" also includes "test compounds".

The term "microRNA" or "miRNA" refers to a single-stranded RNA molecule of at least 10 nucleotides and no more than 35 nucleotides covalently linked together. Preferably, the polynucleotide of the present invention is a molecule of 10 to 33 nucleotides or 15 to 30 nucleotides in length, more preferably 17 to 27 nucleotides or 18 to 26 nucleotides in length, i.e., a molecule of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. The term "microRNA*" or "miRNA*" refers to a miRNA molecule from the passenger strand when processed. In the context of the present invention, the terms "miRNA" and "miRNA*" are used interchangeably. miRBase (www.mirbase.org) is a well-established repository and searchable database of disclosed miRNA sequences and annotations.

The term "whole blood sample" as used in the context of the present invention refers to a blood sample derived from a subject, comprising all blood fractions, including cellular fractions (erythrocytes, leukocytes, platelets) and extracellular blood fractions (serum, plasma). A whole blood sample can be obtained by removing blood from a subject using conventional blood collection techniques, but can also be provided by using a previously separated and/or stored blood sample. Preferably, the volume of a whole blood sample from a subject (such as a human or animal) is 0.1 to 20 ml, more preferably 0.5 to 15 ml, more preferably 1 to 10 ml, and most preferably 2 to 7.5 ml, i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ml. Preferably, the whole blood sample is collected by blood collection tubes, preferably in PAXgene blood RNA tubes, Tempus blood RNA tubes, EDTA tubes (e.g., K2-EDTA Monovette tubes), sodium citrate (Na-citrate) tubes, heparin-tubes, or ACD (citrate dextrose) tubes. Preferably, when collecting whole blood samples, the RNA fraction, in particular the miRNA fraction, can be protected/guarded to prevent degradation. For this purpose, specific collection tubes (e.g., PAXgene blood RNA tubes from Preanalytix, Tempus blood RNA tubes from Applied Biosystems) or additives that stabilize the RNA fraction and/or miRNA fraction (e.g., RNAlater from Ambion, RNAsin from Promega) can be used.

The term "blood cell sample" used in the context of the present invention refers to a preparation of a whole blood sample comprising or substantially comprising blood cells (erythrocytes, leukocytes, platelets), more preferably, a blood cell sample comprising erythrocytes, leukocytes and platelets. Preferably, the blood cell sample does not contain miRNA derived from the extracellular fraction (e.g., plasma, serum) of whole blood or it contains a small amount of miRNA derived from the extracellular fraction (e.g., plasma, serum) such that these do not or substantially do not contribute to the expression profile of the group of one or more miRNAs in the blood cell sample according to the present invention. A blood cell sample comprising erythrocytes, leukocytes and/or platelets or a blood cell sample containing erythrocytes, leukocytes and platelets is obtained from the processing of a whole blood sample collected in a PAXgene blood RNA tube, a Tempus blood RNA tube, an EDTA-tube (e.g., a K2-EDTA Monovette tube), a sodium citrate tube or a heparin tube, maintaining or substantially maintaining the initial cell distribution (blood cell composition) of the whole blood sample. Total RNA (including a short RNA fraction, which includes a miRNA fraction) is isolated from a blood cell sample and used to determine the expression profile of a panel of at least one, preferably at least two, miRNAs in a sample from a subject.

The term "blood cell fraction" as used herein relates to a cell fraction from a blood sample, i.e. a blood cell fraction. The blood cell fraction includes a subfraction (i.e., T cell, B cell, NK cell, neutrophil, granulocyte, reticulocyte, etc.) of red blood cells, white blood cells, platelets or the aforementioned blood cell types or a combination from the aforementioned entities. Preferably, the blood cell fraction does not contain miRNA derived from the extracellular fraction (e.g., blood plasma, serum) of whole blood or it contains a small amount of miRNA derived from the extracellular fraction (e.g., blood plasma, serum) such that these do not or substantially do not contribute to the expression profile of the group of one or more miRNA in the blood cell fraction according to the present invention. In the context of the present invention, the terms "blood cell sample" and "sample of blood cell fraction" are used interchangeably.

The term "total RNA" as used herein relates to isolated RNA that is included in the miRNA fraction present in each hemocyte sample from a whole blood sample. Preferably, the total RNA according to the present invention contains a miRNA fraction or a miRNA-rich fraction containing the total RNA. Total RNA (comprising a miRNA fraction or a miRNA-rich fraction) is obtained by: hemocytes in a lysis (e.g., Trizol) hemocyte sample, followed by RNA purification, e.g., by phenol/chloroform extraction and/or based on separation techniques (e.g., glass fiber filter column, silica membrane column). Examples of kits for RNA separation and purification include: miRNeasy kit (Qiagen), PAXgene blood miRNA kit (Qiagen), mirVana PARIS kit (Life Technologies), PARIS kit (Life Technologies), Tempus Spin RNA isolation kit (Life Technologies).

As used herein, the term "disease-regulated miRNA" refers to at least one fixed defined miRNA contained in a (regulated) group that is known to differ between healthy and diseased subjects. Preferred disease-regulated miRNAs are selected from the miRNAs listed in Figure 1, i.e., miRNAs having SEQ ID NOs: 1 to 26.

As used herein, the term "disease-maintaining miRNA" refers to at least one fixed, defined miRNA that is known to be present in a (unregulated) group that is indistinguishable between healthy and diseased subjects. Preferred disease-maintaining miRNAs are selected from the group consisting of the miRNAs listed in Figure 2, i.e., miRNAs having SEQ ID NOs: 27 to 59.

As used herein, the term "cancer-regulated miRNA" refers to at least one fixed defined miRNA comprised in a (regulated) panel known to be differentially expressed between healthy subjects and subjects suffering from cancer. Preferred cancer-regulated miRNAs are selected from the miRNAs listed in FIG3 .

As used herein, the term "cancer-maintained miRNA" refers to at least one fixed defined miRNA that is known to be included in an (unregulated) group that is indistinguishable between healthy subjects and subjects suffering from cancer. Preferred cancer-maintained miRNAs are selected from the miRNAs listed in FIG4 .

The term " expression profile " used in the context of the present invention refers to the mensuration of miRNA expression profile, or refers to the measurement relevant to the expression of miRNA in sample (for example, from the hemocyte sample or the hemocyte fraction of whole blood sample).By measuring miRNA expression profile, each miRNA is represented by numerical value.The value of independent miRNA is higher, and the expression level of described miRNA is higher, or the value of independent miRNA is lower, and the expression level of described miRNA is lower.Expression profile can be produced by allowing the miRNA expression profile in analysis object and any convenient means comparing between sample, for example, nucleic acid hybridization (for example, to microarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), for quantitative ELISA, next generation sequencing (for example ABISOLID, Illumina genome analyzer, Roche/454GS FLX), flow cytometry (for example LUMINEX, MiliporeGuava) etc. The sample material measured by the aforementioned means is from a blood cell sample and can be total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cDNA, miRNA, labeled miRNA, amplified miRNA, or any derivative that can be generated from the aforementioned RNA/DNA materials. As used herein, an “expression profile” relates to one or more miRNAs, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 51, 53, 54, 55, 56, 57, 58, 59 or more miRNAs, preferably selected from the group consisting of miRNAs having SEQ ID NOs: 1 to 77, more preferably from the group consisting of expression profiles of miRNAs having SEQ ID NOs: 1 to 59.

As used herein, the term "determining an expression profile in (or from) a blood cell sample" relates to determining an expression profile from miRNA present in the blood cell sample, and is therefore a measure related to the miRNA present in the blood cell sample. This includes all steps and transformations required to render the blood cell sample in a form that allows recording of the expression profile by any convenient means (e.g., nucleic acid hybridization, nucleic acid amplification, polymerase extension, mass spectrometry, flow cytometry, sequencing, next generation sequencing) and known to those skilled in the art, such as cell lysis, RNA isolation, RNA labeling, RNA polymerase extension, reverse transcription of RNA ligation to cDNA, cDNA amplification, cDNA labeling, etc.

The term "diagnosis" as used in the context of the present invention refers to the process of determining or monitoring a possible disease or condition, and is therefore a process that attempts to define or monitor the health status of a subject. Determination of the expression profile of one or more miRNAs selected from a list of disease-modulating ( FIG. 1 ; SEQ ID NOs: 1 to 26) and one or more disease-maintaining miRNAs ( FIG. 2 , SEQ ID NOs: 27 to 59) is correlated with the health status of the subject. Nucleic acid hybridization can be performed using arrays/biochips or in situ hybridization. Nucleic acid amplification can be performed using real-time polymerase chain reaction (RT-PCR), such as real-time quantitative polymerase chain reaction (RT qPCR). The aforementioned real-time polymerase chain reaction (RT-PCR) may include the following steps: (i) extracting total RNA from a blood cell sample of a whole blood sample from a subject, (ii) obtaining a cDNA sample by RNA reverse transcription (RT) reaction using universal or miRNA-specific RT (reverse transcription) primers (e.g., stem-loop RT primers); (iii) optionally amplifying the obtained cDNA (e.g., by PCR, such as specific target amplification (STA); (iv) (real-time) quantification of the cDNA in step (ii) or (iii), for example, by real-time polymerase chain reaction to detect the level of miRNA in the sample, wherein a fluorescent dye (e.g., SYBR Green) or a fluorescent probe (e.g., Taqman probe) is added. In step (i), when RT-PCR is performed directly on a sample containing miRNA, RNA separation and/or extraction may be omitted. Kits for determining miRNA expression profiles by real-time polymerase chain reaction (RT-PCR) are available from, for example, Life Technologies, Applied Biosystems, and others. Biosystems, Ambion, Roche, Qiagen, Invitrogen, SABiosciences, Exiqon. In step (ii), the universal or miRNA-specific RT primer can be a single-stranded (DNA) oligonucleotide attached to the 3'-end of the miRNA, thereby generating a non-naturally occurring miRNA-DNA chimera molecule, which is transcribed into DNA by reverse transcriptase.

Nucleic acid sequencing can be performed by conventional Sanger sequencing or by so-called "next generation sequencing (NGS)", which includes but is not limited to sequencing by synthesis, sequencing by ligation, single molecule sequencing, nanopore sequencing or similar technologies. NGS may include the following steps: (i) extracting total RNA from a blood cell sample of a whole blood sample from a subject, (ii) obtaining a cDNA sample by RNA reverse transcription (RT) reaction using universal or miRNA-specific RT (reverse transcription) primers (e.g., stem-loop RT primers), (iii) optionally amplifying the obtained cDNA transcripts (e.g., by PCR), (iv) subjecting the miRNA cDNA transcripts to the aforementioned sequencing techniques, such as sequencing by synthesis or sequencing by ligation. In step (ii), a single-stranded oligonucleotide is ligated to the 3'-end containing a universal or miRNA-specific reverse transcriptase (RT) primer. In step (iii), if the cDNA is to be amplified, the oligonucleotide ligated to the 3'-end also contains a PCR primer sequence, and the single-stranded oligonucleotide is also ligated to the 5'-end, which also contains a PCR primer sequence. Then, in step (iii), the cDNA is amplified using the primer sequences introduced at the 3' and 5'-ends in step (ii).

The term "health status" as used in the context of the present invention relates to a measure of the overall health status of a subject, including but not limited to assessing the presence or absence of a disease or illness in the subject. According to the present invention, the health status of a subject can be assessed by measuring a panel of one or more disease-modulating miRNAs in a blood sample from the subject, preferably a blood cell sample or a blood cell fraction, in combination with a panel of one or more disease-sustaining miRNAs. The health status can be the presence or absence of a disease in the subject, and/or the presence or absence of cancer in the subject. Using the methods of the present invention, changes in the health status are diagnosed. The change in the health status can be an improvement or worsening of the health status, for example, an improvement or worsening of a disease, and/or an improvement or worsening of cancer.

The term "disease" as used herein refers to an abnormal condition that affects the body of a subject. Disease is generally interpreted as a medical condition associated with specific symptoms and signs. Disease can be caused by factors from an external source, such as an infectious disease, or it can be caused by internal dysfunction, such as an autoimmune disease. In humans, "disease" is generally more broadly used to refer to any condition that causes pain, dysfunction, grief, social problems or death in the afflicted subject, or similar problems in people who come into contact with the subject. In a broader sense, it sometimes includes injury, disability, illness, syndrome, infectious disease, isolated symptoms, abnormal behavior, and atypical changes in structure and function, but in other contexts and for other purposes, these can be considered to be distinguishable categories. Disease generally affects the subject not only physically, but also emotionally, because contracting many diseases and living with them can change a person's perspective on life and a person's personality. Cancer is also encompassed by the term "disease" as used herein.

The term "infectious disease" as used herein refers to any disease that can be transmitted from subject to subject and caused by a microbial agent (e.g., a cold). Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia infection or gonorrhea), tuberculosis, HIV/acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

The term "autoimmune disease" as used herein refers to any disease in which the body produces immunogenic (i.e., immune system) responses to certain components of its own tissues. In other words, the immune system has lost the ability to identify some tissues or systems in the body as self, and targets and attacks the tissues or systems as if they were exogenous. Autoimmune diseases can be classified as: diseases (such as hemolytic anemia and anti-immune thyroiditis) in which a major organ is affected, and diseases (such as systemic lupus erythematosus) in which the autoimmune disease process is spread by many tissues. For example, multiple sclerosis is considered to be caused by T cell attacks on the sheaths of the nerve fibers around the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art, and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, etc.

As used herein, the term "cancer disease" or "cancer" refers to or describes a physiological condition in a subject that is typically characterized by dysregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancers include: bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal cord axis tumors, gliomas, meningiomas, and pituitary adenomas. According to the present invention, the term "cancer" also includes cancer metastasis.

The term "subject," as used in the context of the present invention, refers to an individual whose health status is to be assessed. Thus, a subject may be afflicted with or not afflicted by a disease and/or cancer, or may be suspected of being afflicted with or not afflicted by a disease and/or cancer. Using the methods of the present invention, a change in health status is diagnosed. The change in health status may be an improvement or deterioration in health status, for example, an improvement or deterioration in a disease and/or an improvement or deterioration in cancer.

The term "therapeutic treatment" refers to any treatment that improves the health status and/or prolongs (increases) the life span of a subject. The treatment may eliminate the disease in the subject, prevent or slow the progression of the disease in the subject, inhibit or slow the progression of the disease in the subject, reduce the frequency or severity of symptoms in the subject, and/or reduce recurrence in a subject currently suffering from or previously suffering from the disease.

Embodiments of the invention

The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless otherwise indicated, each aspect defined in this manner may be combined with any other aspect. Specifically, unless otherwise indicated, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

In a first aspect, the present invention relates to a method for diagnosing a health condition of a subject, comprising the steps of:

(a) determining the expression profile of a first predetermined set of one or more (disease-modulating) miRNAs in a blood sample from a subject, wherein the miRNAs included in the first predetermined set of miRNAs are selected from the miRNAs listed in Figure 1 and/or Figure 3,

(b) determining the expression profile of a second predetermined set of one or more (disease-maintaining) miRNAs in said blood sample from said subject, wherein the miRNAs included in said second predetermined set of miRNAs are selected from the miRNAs listed in Figures 2 and/or 4,

(c) normalizing the expression profile of the first predetermined group of miRNAs to that of the second predetermined group of miRNAs,

(d) comparing the normalized expression profile from step (c) with a reference,

(e) diagnosing a change in the subject's health condition when the comparison with the reference changes.

Here, the blood sample is selected from the group consisting of whole blood, blood cells, a blood cell fraction consisting of red blood cells, white blood cells, and platelets, a blood cell fraction containing red blood cells, white blood cells, and platelets, or a blood cell fraction containing red blood cells, white blood cells, or platelets. Preferably, the blood sample is a blood cell sample, more preferably, it is a blood cell fraction containing red blood cells, white blood cells, and platelets.

According to the present invention, a whole blood sample is collected from a subject by conventional blood drawing techniques. Blood collection tubes suitable for collecting whole blood include EDTA-tubes (e.g., K2-EDTA Monovette tubes), sodium citrate-tubes, ACD-tubes, heparin-tubes, PAXgene blood RNA-tubes, and Tempus blood RNA-tubes. According to the present invention, the collected whole blood sample (which can be stored immediately before use) is processed to produce a blood cell sample of whole blood. This can be achieved by separating the blood cell fraction (the cellular fraction of whole blood) from the serum/plasma fraction (the extracellular fraction of whole blood), such as by centrifugation. Preferably, the blood cell sample from the whole blood sample comprises red blood cells, white blood cells, and/or platelets, and more preferably, the blood cell sample from the whole blood sample comprises red blood cells, white blood cells, and platelets.

Preferably, total RNA is separated from the blood cells present in the blood cell sample, including miRNA fractions, or miRNA fractions. The kit for separating total RNA (including miRNA fractions) or the kit for separating miRNA fractions are well known to those skilled in the art, such as, miRNeasy kit (Qiagen, Hilden, Germany), Paris kit (Life Technologies, Weiterstadt, Germany). Then, the miRNA (selected from the miRNA listed in FIG. 1 with SEQ ID NO: 1 to 26) and the miRNA (selected from the miRNA listed in FIG. 2 with SEQ ID NO: 27 to 59) of the first group of one or more disease-regulated RNAs separated from the blood cell sample present in whole blood are measured. Expression profile can be determined by any convenient means for measuring miRNA or miRNA spectrum. Various techniques are well known to those skilled in the art, such as, based on the technology or combination of the above-mentioned techniques of nucleic acid hybridization, nucleic acid amplification, sequencing, mass spectrometry, flow cytometry.

Preferably, the normalization in step (c) produces a relative expression level of each miRNA contained in the first predetermined group of miRNAs. Preferably, the relative expression level is a ratio obtained from the ratio of one or more miRNAs contained in the first group of (disease- and/or cancer-regulated) miRNAs from one or more subjects to be diagnosed to the ratio of one or more miRNAs contained in the second group of (disease- and/or cancer-maintained) miRNAs. More preferably, the relative expression level is obtained by dividing the expression level of one or more miRNAs contained in the first group of (disease- and/or cancer-regulated) miRNAs by the level of one or more miRNAs contained in the second group of (disease- and/or cancer-maintained) miRNAs. Thus, the second group of (disease- and/or cancer-maintained) miRNAs is used as a standard for the first group of (disease- and/or cancer-regulated) miRNAs. Examples of such relative expression levels are ratios of the following miRNA expression levels from one or more subjects to be diagnosed: hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-425 (SEQ ID NO: 36), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-425-3p (SEQ ID NO: 37), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-425-3p (SEQ ID NO: 38). NO:1) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-140-3p (SEQ ID NO:2 NO:35), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-2 NO:3) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-92a (SEQ ID NO:41). NO:27), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:39), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:40), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:41), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 NO:5) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-30b (SEQ ID NO: NO:37), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140 NO:7) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-19a (SEQ ID NO: NO:29), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-126 (SEQ ID NO:8) divided by hsa-miR-92b (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:8) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-92c (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:8) divided by hsa-miR-1 NO:9) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-140-3p (SEQ ID NO:35). NO:39), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:37 ...8), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:38), or hsa-miR-20a (SEQ ID NO:10) divided by NO:10) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-425 (SEQ ID NO:11) hsa-miR-374b (SEQ ID NO: 11) divided by hsa-miR-30b (SEQ ID NO: 37), or hsa-miR-374b (SEQ ID NO: 11) divided by hsa-miR-22 (SEQ ID NO: 38), or hsa-miR-374b (SEQ ID NO: 11) divided by hsa-miR-25 (SEQ ID NO: 39), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-126 (SEQ ID NO: 9) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO: 39), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-126 (SEQ ID NO: 9) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO: 39), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-20b (SEQ ID NO: 39), or hsa-miR-144 NO:6) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-192 (SEQ ID NO:41), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-192 (SEQ ID NO:42). NO:41), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-874 (SEQ ID NO:59), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO: NO:8) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-27b (SEQ ID NO:34), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-222 (SEQ ID NO:15) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20b (SEQ ID NO:34). NO:6) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-99b (SEQ ID NO:54), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-361-3p (SEQ ID NO:55).

Preferably, the reference to which the normalized expression profile is compared in step (d) is a reference expression profile. Preferably, when obtaining an expression profile from a subject to be diagnosed, the reference expression profile is obtained in a comparable manner from one or more reference subjects. Preferably, the reference is a normalized reference expression profile obtained by comprising the steps of:

(i) determining the expression profile of a first predetermined group of one or more miRNAs in a blood sample from one or more reference subjects, wherein the miRNAs included in the first predetermined group of miRNAs are selected from the miRNAs listed in Figure 1 and/or Figure 3,

(ii) determining the expression profile of a second predetermined set of one or more miRNAs in the blood sample from one or more reference subjects, wherein the miRNAs included in the second predetermined set of miRNAs are selected from the miRNAs listed in Figure 2 and/or Figure 4,

(iii) normalizing the expression profiles of the first predetermined set of miRNAs in the one or more reference subjects to the second predetermined set of miRNAs.

It is also preferred that the normalization in step (iii) produces a relative reference expression level of each miRNA contained in the first predetermined group of miRNAs. Preferably, the relative reference expression level is a ratio of one or more miRNAs contained in the first group of (disease- and/or cancer-regulated) miRNAs obtained from one or more reference subjects to the ratio of one or more miRNAs contained in the second group of (disease- and/or cancer-maintained) miRNAs. More preferably, the relative expression level is obtained by dividing the expression level of one or more miRNAs contained in the first group of (disease- and/or cancer-regulated) miRNAs from one or more reference subjects by the level of one or more miRNAs contained in the second group of (disease- and/or cancer-maintained) miRNAs. Thus, the second group of (disease- and/or cancer-maintained) miRNAs is used as a standard for the first group of (disease- and/or cancer-regulated) miRNAs. Examples of such relative reference expression levels are ratios of the following miRNA expression levels from one or more reference subjects: hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-19a (SEQ ID NO: 29), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-140-3p (SEQ ID NO: 35), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-425 (SEQ ID NO: 36), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-425-3p (SEQ ID NO: 37), or hsa-miR-423-5p (SEQ ID NO: 1) divided by hsa-miR-425-3p (SEQ ID NO: 38). ID NO:1) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-423-5p (SEQ ID NO:1) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-140-3p (SEQ ID NO:2 NO:35), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-26b (SEQ ID NO:2) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-2 NO:3) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-374a (SEQ ID NO:3) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-92a (SEQ ID NO:41). NO:27), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:39), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:40), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 (SEQ ID NO:41), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-22 NO:5) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-30b (SEQ ID NO: NO:37), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140 NO:7) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-19a (SEQ ID NO: NO:29), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-126 (SEQ ID NO:8) divided by hsa-miR-92b (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:8) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-92c (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:8) divided by hsa-miR-1 NO:9) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-126 (SEQ ID NO:9) divided by hsa-miR-25 (SEQ ID NO:39). NO:39), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-425 (SEQ ID NO:36), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-19a (SEQ ID NO:29), NO:10) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-25 (SEQ ID NO:39), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-374b (SEQ ID NO:11) divided by hsa-miR-425 (SEQ ID NO:11) hsa-miR-374b (SEQ ID NO: 11) divided by hsa-miR-30b (SEQ ID NO: 37), or hsa-miR-374b (SEQ ID NO: 11) divided by hsa-miR-22 (SEQ ID NO: 38), or hsa-miR-374b (SEQ ID NO: 11) divided by hsa-miR-25 (SEQ ID NO: 39), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-126 (SEQ ID NO: 9) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO: 39), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-92a (SEQ ID NO: 27), or hsa-miR-126 (SEQ ID NO: 9) divided by hsa-miR-16 (SEQ ID NO: 28), or hsa-miR-20b (SEQ ID NO: 39), or hsa-miR-144* (SEQ ID NO: 7) divided by hsa-miR-20b (SEQ ID NO: 39), or hsa-miR-144 NO:6) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-92a (SEQ ID NO:27), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-192 (SEQ ID NO:41), or hsa-miR-20b (SEQ ID NO:6) divided by hsa-miR-192 (SEQ ID NO:42). NO:41), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-874 (SEQ ID NO:59), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-30b (SEQ ID NO:37), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-16 (SEQ ID NO:28), or hsa-miR-17 (SEQ ID NO:8) divided by hsa-miR-20a (SEQ ID NO:10) divided by hsa-miR-140-3p (SEQ ID NO:35), NO:8) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-106a (SEQ ID NO:13) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-27b (SEQ ID NO:34), or hsa-miR-144* (SEQ ID NO:7) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-222 (SEQ ID NO:15) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-20b (SEQ ID NO:34). NO:6) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-22 (SEQ ID NO:38), or hsa-miR-363 (SEQ ID NO:24) divided by hsa-miR-140-3p (SEQ ID NO:35), or hsa-miR-93* (SEQ ID NO:21) divided by hsa-miR-99b (SEQ ID NO:54), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-19a (SEQ ID NO:29), or hsa-miR-720 (SEQ ID NO:5) divided by hsa-miR-361-3p (SEQ ID NO:55).

According to the present invention, the comparison in step (d) is performed using the normalized expression level from the subject to be diagnosed and a normalized reference expression profile from one or more reference subjects, the normalized reference expression profile being obtained by employing the same combination of miRNAs, i.e. the same combination of disease-modulated miRNAs and disease-maintaining miRNAs, more preferably the same ratio of disease-modulated miRNAs and disease-maintaining miRNAs.

Preferably, the comparison in step (d) is by comparing the relative expression level from the subject with a relative reference expression level of each miRNA comprised in the first predetermined set of miRNAs.

Preferably, relative expression levels are obtained by calculating the ratio of the miRNAs listed in FIG1 (disease-modulated) to the miRNAs listed in FIG2 (disease-maintaining).

It is also preferred that the relative expression levels are selected from the ratios listed in FIG. 7 .

It is further preferred that in step (e), a change in health condition is diagnosed when the relative expression level from the subject compared to the relative reference expression level exceeds a threshold value for one or more of said predetermined miRNAs comprised in said panel.

Preferably, the threshold for diagnosing changes in health status is set at 3 standard deviations, preferably 5 standard deviations, and more preferably 7 standard deviations.

In a second aspect, the present invention relates to a method of diagnosing a health condition in a subject.

It should be understood that the method according to the second aspect of the present invention includes and/or comprises the aspects detailed in the method according to the first aspect of the present invention.

In said second aspect, the present invention relates to a method for diagnosing a health condition of a subject, comprising the steps of:

(a) determining the expression profile of a first predetermined set of miRNAs in a blood sample from a subject,

(b) determining the expression profile of a second predetermined set of miRNAs in said blood sample from said subject,

(c) comparing the expression profile of step (a) and the expression profile of step (b) with a reference expression profile,

(d) diagnosing a change in the subject's health condition when the comparison to the first reference expression profile is changed and the comparison to the second reference expression profile is unchanged.

Here, the miRNAs included in the first predetermined group of miRNAs and the miRNAs included in the first reference expression profile are selected from the miRNAs listed in Figures 1 and/or 3, and the miRNAs included in the second predetermined group of miRNAs are selected from the miRNAs listed in Figures 2 and/or 4.

Preferably, the blood sample is selected from whole blood, blood cells, a blood cell fraction consisting of red blood cells, white blood cells and platelets, a blood cell fraction comprising red blood cells, white blood cells and platelets, or a blood cell fraction comprising red blood cells, white blood cells or platelets. More preferably, the blood sample is a blood cell fraction comprising red blood cells, white blood cells and platelets.

Preferably, the reference expression profiles according to the first and second aspects of the present invention are from an earlier time point of the same subject.

Preferably, the change in health condition according to the first and second aspects of the present invention is an improvement or deterioration of health condition.

Preferably, in the method according to the first aspect or the second aspect of the present invention, when the expression profile is determined in a blood cell sample from a whole blood sample, the nucleic acid sequences of at least two miRNAs, i.e., one disease-modulating miRNA (of one or more miRNAs selected from the first predetermined group of miRNAs listed in FIG. 1 having SEQ ID NOs: 1 to 26) and one disease-maintaining miRNA (of one or more miRNAs selected from the second predetermined group of miRNAs listed in FIG. 2 having SEQ ID NOs: 27 to 59) have SEQ ID NO: 1 and SEQ ID NO: 27, the nucleic acid sequences of at least two miRNAs have SEQ ID NO: 1 and SEQ ID NO: 28, the nucleic acid sequences of at least two miRNAs have SEQ ID NO: 1 and SEQ ID NO: 29, the nucleic acid sequences of at least two miRNAs have SEQ ID NO: 1 and SEQ ID NO: 30, the nucleic acid sequences of at least two miRNAs have SEQ ID NO: 1 and SEQ ID NO: 31, the nucleic acid sequences of at least two miRNAs have SEQ ID NO: 1 and SEQ ID NO: 32, the nucleic acid sequences of at least two miRNAs have SEQ ID NO: 1 and SEQ ID NO: 33. NO:33, at least two miRNAs have nucleic acid sequences having SEQ ID NO:1 and SEQ ID NO:34, at least two miRNAs have nucleic acid sequences having SEQ ID NO:1 and SEQ ID NO:35, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:28, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:29, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:30, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:31, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:32, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:33, at least two miRNAs have nucleic acid sequences having SEQ ID NO:2 and SEQ ID NO:34, at least two miRNAs have nucleic acid sequences having SEQ ID NO:3 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO: NO:3 and SEQ ID NO:28, at least two miRNAs have nucleic acid sequences having SEQ ID NO:3 and SEQ ID NO:29, at least two miRNAs have nucleic acid sequences having SEQ ID NO:3 and SEQ ID NO:30, at least two miRNAs have nucleic acid sequences having SEQ ID NO:3 and SEQ ID NO:31, at least two miRNAs have nucleic acid sequences having SEQ ID NO:3 and SEQ ID NO:32, at least two miRNAs have nucleic acid sequences having SEQ ID NO:3 and SEQ ID NO:33, at least two miRNAs have nucleic acid sequences having SEQ ID NO:4 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO:4 and SEQ ID NO:28, at least two miRNAs have nucleic acid sequences having SEQ ID NO:4 and SEQ ID NO:29, at least two miRNAs have nucleic acid sequences having SEQ ID NO:4 and SEQ ID NO:30, at least two miRNAs have nucleic acid sequences having SEQ ID NO:4 and SEQ ID NO:31, at least two miRNAs have nucleic acid sequences having SEQ ID NO:4 and SEQ ID NO: NO:32, at least two miRNAs have nucleic acid sequences having SEQ ID NO:5 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO:5 and SEQ ID NO:28, at least two miRNAs have nucleic acid sequences having SEQ ID NO:5 and SEQ ID NO:29, at least two miRNAs have nucleic acid sequences having SEQ ID NO:5 and SEQ ID NO:30, at least two miRNAs have nucleic acid sequences having SEQ ID NO:5 and SEQ ID NO:31, at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:28, at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:29, at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:30, at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:28, at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:29. NO:7 and SEQ ID NO:29, at least two miRNAs have nucleic acid sequences having SEQ ID NO:8 and SEQ ID NO:27, at least two miRNAs have nucleic acid sequences having SEQ ID NO:8 and SEQ ID NO:29, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:9 and SEQ ID NO:27, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:27, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:9 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:35, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:27, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:24 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:35. NO:38, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:24 and SEQ ID NO:41, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:6 and SEQ ID NO:41, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:5 and SEQ ID NO:59, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:8 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:10 and SEQ ID NO:35, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:10 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:37, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:13 and SEQ ID NO:28, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:8 and SEQ ID NO:35, or at least two miRNAs have nucleic acid sequences having SEQ ID NO:7 and SEQ ID NO:35, or at least two miRNAs have nucleic acid sequences having SEQ ID NO: NO:13 and SEQ ID NO:35, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:21 and SEQ ID NO:34, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:7 and SEQ ID NO:29, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:15 and SEQ ID NO:35, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:6 and SEQ ID NO:29, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:24 and SEQ ID NO:38, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:24 and SEQ ID NO:35, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:21 and SEQ ID NO:54, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:5 and SEQ ID NO:29, or the nucleic acid sequence of at least two miRNAs has SEQ ID NO:5 and SEQ ID NO:55.

Preferably, the expression profile is determined by the steps comprising:

(a) providing a whole blood sample of a subject whose health condition is to be diagnosed,

(b) obtaining a blood cell sample from the whole blood sample,

(c) extracting total RNA from the blood cell sample,

(d) determining the expression profiles of a first set of one or more disease-modulated miRNAs (selected from the miRNAs listed in Figure 1 with SEQ ID NOs: 1 to 26) and a second set of one or more disease-maintaining miRNAs (selected from the miRNAs listed in Figure 2 with SEQ ID NOs: 27 to 59) from the extracted total RNA.

Preferably, a blood cell sample is obtained from a whole blood sample by separating the blood cells from the remaining fraction of the whole blood sample, which can be achieved by centrifugation, wherein the blood cells (including red blood cells, white blood cells and platelets) form a pellet that can be harvested (see Example 1). Those skilled in the art are aware of alternative methods for separating blood cells from the remaining fraction of a whole blood sample (e.g., size exclusion, size distribution, two-dimensional electrophoresis, positive or negative antibody selection, etc.).

It is also preferred that the mensuration of the expression spectrum comprises that at least one miRNA, preferably at least two miRNA nucleotide sequences are reverse transcribed into cDNA (complementary DNA) by the mensuration of the expression spectrum of the miRNA.Herein, before measuring the expression spectrum of the miRNA, the RNA sequence is reverse transcribed into DNA (for example, by using reverse transcriptase).Preferably, when adopting the technology based on nucleic acid amplification (PCR, RT-PCR), order-checking (next generation sequencing, Sanger order-checking) or hybridization in the mensuration of the miRNA expression spectrum, at least one miRNA, preferably at least two miRNA nucleotide sequences are reverse transcribed into cDNA.In addition, it is preferred that total RNA is transcribed into the cDNA measuring expression spectrum.

In another embodiment of the present invention according to the first and second aspects, the disease-regulated miRNA is a cancer-regulated miRNA selected from the miRNAs listed in Figure 3, and the disease-maintained miRNA is a cancer-maintained miRNA selected from the miRNAs listed in Figure 4.

In a third aspect, the present invention relates to the use of the method according to the first and second aspects of the invention.

It should be understood that the use according to the third aspect of the present invention includes and/or comprises the aspects specified in the methods according to the first and second aspects of the present invention.

The third aspect of the present invention relates to the use of the method according to the first and second aspects of the present invention for the following:

(i) comparing the health status of one or more subjects,

(ii) monitoring the health status of the subject,

(iii) monitoring the status of the subject's immune system,

(iv) monitoring the subject's response to therapeutic treatment, preferably pharmaceutical treatment.

In a fourth aspect, the present invention relates to a kit for diagnosing a health condition in a subject, comprising:

(i) a device for determining an expression profile of a group comprising at least one miRNA selected from SEQ ID NOs: 1 to 26 and at least one miRNA selected from SEQ ID NOs: 27 to 59 in a blood sample of a subject, and

(ii) at least one reference.

It should be understood that the kit according to the fourth aspect of the present invention includes and/or comprises the aspects detailed in the methods according to the first and second aspects of the present invention, and the aspects detailed in the use according to the third aspect of the present invention.

Preferably, the apparatus for determining the expression profile comprises:

(i) a set of at least two polynucleotides for measuring a group comprising at least one miRNA selected from SEQ ID NOs: 1 to 26 and at least one miRNA selected from SEQ ID NOs: 27 to 59 in a blood sample of a subject, and

(ii) Arrays, RT-PCR systems, PCR systems, flow cytometry, bead-based multiplex systems, or next-generation sequencing systems.

The kit comprises at least one reference according to the invention as outlined in the first aspect of the invention. In a preferred embodiment, the reference may be contained in a data carrier of the kit. In a further preferred embodiment, the reference may be a reference sample and/or a reference standard, which is contained in the kit and which is employed, for example, when the kit is used in the determination of an expression profile.

Optionally, the kit comprises a data carrier. Preferably, the data carrier is an electronic or non-electronic data carrier, more preferably, it is an electronic data carrier, such as a storage medium. The kit optionally comprises a data carrier, which optionally comprises references and/or instructions on how to use the expression profile and the reference in the diagnosis of the health condition. This instruction on how to use the expression profile and the reference may include instructions for doctors and/or diagnostic laboratories involved in the diagnosis of the health condition. Preferably, the data carrier also comprises tools for analyzing and evaluating the measured expression profile. These tools may be any tools that help doctors and/or diagnostic laboratories diagnose the health condition. Preferably, the instructions comprised are algorithms or software. Preferably, these tools are software tools that help analyze the measured expression profile and/or help with subsequent diagnosis. The tools for analysis and evaluation may include references according to the present invention.

The kit optionally comprises a whole blood collection tube, which is preferably selected from EDTA-tubes, sodium citrate-tubes, ACD-tubes, heparin-tubes, PAXgene blood RNA-tubes, Tempus blood RNA-tubes, and optionally contains additives for stabilizing the RNA fraction.

The kit optionally includes a device for obtaining a blood cell sample from a whole blood sample. These devices are preferably used to separate and/or isolate each blood cell sample (e.g., a blood cell sample comprising white blood cells, red blood cells, or platelets, a blood cell sample comprising white blood cells, red blood cells, and platelets, a platelet preparation) from the remaining fraction of the whole blood sample. These devices may include reagents or consumables and/or suitable instruments (e.g., centrifuges, dedicated collection tubes) for separating/isolating each blood cell fraction.

In another aspect of the present invention, the inventors have found that in blood samples, preferably whole blood samples or blood cell fractions comprising red blood cells, white blood cells and/or platelets, certain miRNAs (cancer-regulated miRNAs, FIG3 ) are significantly deregulated between healthy controls and cancer subjects, whereas certain other miRNAs (cancer-sustaining miRNAs, FIG4 ) are not significantly deregulated between healthy controls and cancer subjects. It will be appreciated that in further embodiments of the present invention, the cancer-regulated miRNAs ( FIG3 ) and the cancer-sustaining miRNAs ( FIG4 ) can be used in the same manner as described in detail for the disease-regulated miRNAs and disease-sustaining miRNAs according to the first, second, third and fourth aspects of the present invention to diagnose a health condition in a subject, wherein a change in health condition relates to the absence or presence of cancer in the subject.

In other aspects, the present invention relates to a method of monitoring the health of a subject, comprising the steps of:

(a) diagnosing the health status of a subject at a first time point by implementing the method according to the first aspect or the second aspect,

(b) diagnosing the health status of the subject at one or more later time points by performing the method according to the first aspect or the second aspect, and

(c) comparing the health condition diagnosed in step (a) with the health condition diagnosed in step (b), thereby monitoring the health condition of the subject.

In one embodiment, the subject's health condition improves or worsens over time.

In one other embodiment, the monitored health condition of the subject encompasses the state of the subject's immune system.

In another embodiment, the subject has received therapeutic treatment between the first time point and one or more later time points. The treatment may be a pharmaceutical treatment, i.e., it may comprise administration of a drug. The drug may be a drug for treating the subject's disease and/or cancer. The treatment may alternatively and/or additionally comprise surgery, chemotherapy, radiotherapy, and/or immunotherapy.

In summary, the present invention consists of the following:

1. A method for diagnosing a health condition of a subject, comprising the steps of:

(f) determining the expression profile of a first predetermined group of one or more miRNAs in a blood sample from the subject, wherein the miRNAs included in the first predetermined group of miRNAs are selected from the miRNAs listed in Figure 1 and/or Figure 3,

(g) determining the expression profile of a second predetermined set of one or more miRNAs in the blood sample from the subject, wherein the miRNAs included in the second predetermined set of miRNAs are selected from the miRNAs listed in Figure 2 and/or Figure 4,

(h) normalizing the expression profile of the first predetermined group of miRNAs to that of the second predetermined group of miRNAs,

(i) comparing the normalized expression profile in step (c) with a reference,

(j) diagnosing a change in the subject's health condition when the comparison with the reference changes,

The blood sample is selected from whole blood, blood cells, a blood cell fraction consisting of red blood cells, white blood cells and platelets, a blood cell fraction containing red blood cells, white blood cells and platelets, or a blood cell fraction containing red blood cells, white blood cells or platelets.

2. The method of item 1, wherein the normalization in step (c) produces a relative expression level of each miRNA contained in the first predetermined set of miRNAs.

3. The method of item 2, wherein the reference is a normalized reference expression profile obtained by the steps of:

(i) determining the expression profile of a first predetermined group of one or more miRNAs in a blood sample from one or more reference subjects, wherein the miRNAs included in the first predetermined group of miRNAs are selected from the miRNAs listed in Figure 1 and/or Figure 3,

(ii) determining the expression profile of a second predetermined set of one or more miRNAs in the blood sample from one or more reference subjects, wherein the miRNAs included in the second predetermined set of miRNAs are selected from the miRNAs listed in Figure 2 and/or Figure 4,

(iii) normalizing the expression profiles of the first predetermined set of miRNAs in the one or more reference subjects to the second predetermined set of miRNAs.

4. The method of item 3, wherein the normalization in step (iii) produces a relative reference expression level of each miRNA contained in the first predetermined set of miRNAs.

5. The method of any one of items 1 to 4, wherein the comparison in step (d) is by comparing the relative expression level from the subject with a relative reference expression level of each miRNA contained in the first predetermined set of miRNAs.

6. The method of any one of items 2, 4 or 5, wherein the relative expression levels are obtained by calculating the ratio of the (disease-regulated) miRNAs listed in FIG1 to the (disease-maintaining) miRNAs listed in FIG2.

7. The method of item 6, wherein the relative expression levels are selected from the ratios listed in FIG7 .

8. The method of any one of items 1 to 7, wherein in step (e), a change in health condition is diagnosed when the relative expression level from the subject compared to the relative reference expression level exceeds a threshold value for one or more of the predetermined miRNAs included in the group.

9. The method according to any one of items 1 to 8, wherein the threshold is set to 3 standard deviations, preferably 5 standard deviations, and more preferably 7 standard deviations.

10. A method for diagnosing a health condition of a subject, comprising the steps of:

(e) determining the expression profile of a first predetermined set of miRNAs in a blood sample from the subject,

(f) determining the expression profile of a second predetermined set of miRNAs in said blood sample from said subject,

(g) comparing the expression profile of step (a) and the expression profile of step (b) with a reference expression profile,

(h) diagnosing a change in the subject's health condition when the comparison to the first reference expression profile is changed and the comparison to the second reference expression profile is unchanged,

in

(i) the miRNAs included in the first predetermined group of miRNAs and the miRNAs included in the first reference expression profile are selected from the miRNAs listed in Figure 1 and/or Figure 3, and

(ii) the miRNAs included in the second predetermined group of miRNAs are selected from the miRNAs listed in Figure 2 and/or Figure 4, and

(iii) wherein the blood sample is selected from whole blood, blood cells, a blood cell fraction consisting of red blood cells, white blood cells and platelets, a blood cell fraction comprising red blood cells, white blood cells and platelets, or a blood cell fraction comprising red blood cells, white blood cells or platelets.

11. The method of any one of items 3 to 10, wherein the reference expression profile is from an earlier time point of the same subject.

12. The method according to any one of items 1 to 11, wherein the change in health condition is an improvement or a deterioration in health condition.

13. Use of the method according to any one of items 1 to 12 for:

(i) comparing the health status of one or more subjects,

(ii) monitoring the health status of the subject,

(iii) monitoring the status of the subject's immune system,

(iv) monitoring the subject's response to therapeutic treatment, preferably pharmaceutical treatment.

14. The method, use or kit of any one of items 1 to 13, wherein the additional miRNAs in the first group of (disease-modulating) miRNAs are selected from the miRNAs listed in Figure 3, and wherein the additional miRNAs in the second group of (disease-maintaining) miRNAs are selected from the miRNAs listed in Figure 3, and wherein the change in health status relates to the absence or presence of cancer in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Overview of disease-regulated miRNAs (SEQ ID NOs: 1 to 26) measured in blood cell samples containing red blood cells, white blood cells, and platelets from whole blood samples collected in PAXgene blood RNA tubes. Here, blood samples were drawn into PAXgene blood RNA tubes, total RNA from blood cells (including the blood cell miRNA fraction) was isolated using the miRNeasy kit (http://www.qiagen.com), and analyzed on DNA microarrays (febitbiomed) representing miRBase version 12 (Experimental details: SEQ ID NO: sequence identification number; miRNA: miRNA annotation according to miRBase version 12; median g1(healthy control) = median expression level in healthy control patients; median g2(diseased) = median expression level in diseased subjects; qmedian = ratio of median g1(healthy control) to median g2(diseased); ttest_adjp = Benjamini-Hochberg-corrected p-value calculated according to the T-test; AUC = area under the curve statistic).

FIG2 : Overview of disease-maintaining miRNAs (SEQ ID NOs: 27 to 59) measured in blood cell samples containing red blood cells, white blood cells, and platelets from whole blood samples collected in PAXgene blood RNA tubes. Here, blood samples were drawn into PAXgene blood RNA tubes, and total RNA from blood cells (including the blood cell miRNA fraction) was isolated using the miRNeasy kit (http://www.qiagen.com) and analyzed on DNA microarrays (febitbiomed) representing miRBase version 12 (Experimental details: SEQ ID NO: sequence identification number; miRNA: miRNA annotation according to miRBase version 12; median g1(healthy control) = median expression level in healthy control patients; median g2(diseased) = median expression level in diseased subjects; qmedian = ratio of median g1(healthy control) to median g2(diseased); ttest_adjp = Benjamini-Hochberg-corrected p-value calculated according to the T-test; AUC = area under the curve statistic).

Figure 3: Overview of cancer-regulated miRNAs measured in blood cell samples containing red blood cells, white blood cells, and platelets from whole blood samples collected in PAXgene blood RNA tubes. Here, blood samples were aspirated into PAXgene blood RNA tubes, total RNA from blood cells (including the miRNA fraction of blood cells) was isolated using the miRNeasy kit (http://www.qiagen.com) and analyzed on DNA microarrays (febit biomed) representing miRBase version 12 (Experimental details: SEQ ID NO: sequence identification number; miRNA: miRNA annotation according to miRBase version 12; median g1(healthy control) = median expression level of healthy control patients; median g2(cancer) = median expression level of diseased subjects; qmedian = ratio of median g1(healthy control) to median g2(cancer); ttest_adjp = Benjamini-Hochberg-corrected p-value calculated according to the T-test; AUC = area under the curve statistic).

FIG4 : Overview of cancer-maintained miRNAs measured in blood cell samples containing red blood cells, white blood cells, and platelets from whole blood samples collected in PAXgene blood RNA tubes. Here, blood samples were aspirated into PAXgene blood RNA tubes, and total RNA from blood cells (including the miRNA fraction of blood cells) was isolated using the miRNeasy kit (http://www.qiagen.com) and analyzed on DNA microarrays (febit biomed) representing miRBase version 12 (Experimental details: SEQ ID NO: sequence identification number; miRNA: miRNA annotation according to miRBase version 12; median g1(healthy control) = median expression level of healthy control patients; median g2(cancer) = median expression level of diseased subjects; qmedian = ratio of median g1(healthy control) to median g2(cancer); ttest_adjp = Benjamini-Hochberg-corrected p-value calculated according to the T-test; AUC = area under the curve statistic).

Figure 5: Schematic depiction of a method according to (a) the second aspect of the invention and (b) the first aspect of the invention.

Figure 6: Graphical description of the application of the method according to the invention for monitoring the health status of a subject.

FIG7 : Box plots depicting relative expression levels obtained by normalizing the expression profiles of a first predetermined group of one or more miRNAs (selected from the disease-modulating miRNAs of FIG1 ) to a second predetermined group of miRNAs (selected from the disease-maintaining miRNAs of FIG2 ). Here, the gray box plots on the left represent relative expression levels in healthy control subjects (n=67), and the white box plots on the right represent relative expression levels in diseased subjects (n=713); where pval = Benjamini-Hochberg-corrected p-value calculated from a T-test. Here, miRNAs from blood cell fractions comprising red blood cells, white blood cells, and platelets were used; all samples were collected and processed according to Examples 1-5.

Among them: Figure 7-1: hsa-miR-144*/hsa-miR-16; Figure 7-2: hsa-miR-144*/hsa-miR-92a; Figure 7-3: hsa-miR-126/hsa-miR-16; Figure 7-4: hsa-miR-20b/hsa-miR-16; Figure 7-5: hsa-miR-20b/hsa-miR-140-3p; Figure 7-6: hsa-miR-20b/hsa-miR-92a; Figure 7-7: hsa-miR-363/hsa-miR-16; Figure 7-8: h sa-miR-20b/hsa-miR-22; Figure 7-9: hsa-miR-363/hsa-miR-192; Figure 7-10: hsa-miR-20b/hsa-miR-192; Figure 7-11: hsa-miR-720/hsa-miR- 874; Figure 7-12: hsa-miR-17/hsa-miR-16; Figure 7-13: hsa-miR-20a/hsa-miR-140-3p; Figure 7-14: hsa-miR-20a/hsa-miR-16; Figure 7-15: hsa-miR -144*/hsa-miR-30b; Figure 7-16: hsa-miR-106a/hsa-miR-16; Figure 7-17: hsa-miR-17/hsa-miR-140-3p; Figure 7-18: hsa-miR-144*/hsa-miR-140-3p; Figure 7-19: hsa-miR-106a/hsa-miR-140-3p; Figure 7-20: hsa-miR-93*/hsa-miR-27b; Figure 7-21: hsa-miR-144*/hsa-miR-19a; Figure 7-2 2: hsa-miR-222/hsa-miR-140-3p; Figure 7-23: hsa-miR-20b/hsa-miR-19a; Figure 7-24: hsa-miR-363/hsa-miR-22; Figure 7-25: hsa-miR-363/h sa-miR-140-3p; Figure 7-26: hsa-miR-93*/hsa-miR-99b; Figure 7-27: hsa-miR-720/hsa-miR-19a; Figure 7-28: hsa-miR-720/hsa-miR-361-3p.

(In the figure, annotations of miRNA identifiers are truncated due to visualization constraints; here, relative expression values are listed as above)

Figure 8: Overview of relative expression levels obtained from normalizing the expression profiles of a first predetermined set of one or more miRNAs (selected from the disease-modulated miRNAs of Figure 1) to a second predetermined set of miRNAs (selected from the disease-maintaining miRNAs of Figure 2). (Experimental details: miRNA = miRNA annotation according to miRBase version 12; median ratio r/p(healthy control) = median relative expression level of the healthy control group, where the median is calculated as the ratio of the expression levels of disease-regulated miRNA (r) and disease-maintained miRNA (p) in the healthy control group; median ratio r/p(disease) = median relative expression level of the disease group, where the median is calculated as the ratio of the expression levels of disease-regulated miRNA (r) and disease-maintained miRNA (p) in the disease group; fold change = median fold change in median relative expression level between healthy control and disease group; Wilcoxon MannWhitney test-corrected p-value = corrected p-value calculated according to the WMW test; t-test corrected p-value = Benjamini-Hochberg-corrected p-value calculated according to the T-test; AUC = area under the curve statistic.)

Figure 9: Overview of relative expression levels obtained from normalizing the expression profiles of a first predetermined set of one or more miRNAs (selected from the cancer-regulated miRNAs of Figure 3) to a second predetermined set of miRNAs (selected from the cancer-maintained miRNAs of Figure 4). (Experimental details: miRNA = miRNA annotation according to miRBase version 12; median ratio r/p(healthy control) = median relative expression level of the healthy control group, where the median is calculated as the ratio of the expression levels of cancer-regulated miRNA (r) and cancer-maintained miRNA (p) in the healthy control group; median ratio r/p(cancer) = median relative expression level of the cancer group, where the median is calculated as the ratio of the expression levels of cancer-regulated miRNA (r) and cancer-maintained miRNA (p) in the cancer group; fold change = median fold change of median relative expression level between healthy control and cancer groups; Wilcoxon MannWhitney test-corrected p-value = corrected p-value calculated according to the WMW test; t-test corrected p-value = Benjamini-Hochberg-corrected p-value calculated according to the T-test; AUC = area under the curve statistic.

Figure 10: Classification performance of a predetermined panel of 5 disease-regulated miRNAs and 5 disease-maintaining miRNAs for diagnosing health conditions in subjects. A panel consisting of 5 normalized expression profiles classified health conditions (healthy controls versus disease) with an AUC of 0.78. Classification was performed using 5 normalized expression profiles calculated from hsa-miR-194/hsa-miR-223, hsa-miR-192/hsa-miR-223, hsa-miR-19a/hsa-miR-223, hsa-miR-99b/hsa-miR-484, and hsa-miR-99b/hsa-miR-19b.

Example

The examples are designed to further illustrate the present invention and provide a better understanding. They should not be interpreted as limiting the scope of the present invention in any way.

Example 1: Obtaining a blood cell sample from a whole blood sample

Blood from healthy controls and diseased subjects was collected in PAXgene blood RNA tubes (PreAnalytix). For each blood donor, 2.5 ml of blood was placed in a PAXgene blood RNA tube. A blood cell preparation was obtained by centrifugation of the whole blood sample. Here, blood cells from whole blood collected in a blood collection tube were centrifuged at 5000 x g for 10 min. The blood cell pellet (the blood cell fraction containing red blood cells, white blood cells, and platelets) was harvested for further processing, while the supernatant (including the extracellular blood fraction) was discarded. Total RNA, including small RNA (miRNA fraction), was extracted from the harvested blood cells using the miRNeasy micro kit (Qiagen GmbH, Hilden, Germany); see Example 2 for details.

Example 2: Isolation of total RNA (including microRNA)

Isolation of total RNA (including small RNA (miRNA fraction)) was performed by using the miRNeasy micro kit (Qiagen GmbH, Hilden, Germany). Here, the blood cell pellet (obtained as outlined in Example 1) was thoroughly suspended in 700 μl of QIAzol lysis reagent by pipetting up and down, and the suspension was immediately transferred to a new 1.5 ml Eppendorf tube. Then, 140 μl of chloroform was added, vortexed thoroughly and incubated at room temperature for 2-3 min, followed by centrifugation at 12,000 g for 15 min at 4°C. The upper aqueous phase was then carefully transferred to a new 2 ml tube without touching the other two phases. Then, 1.5 volumes of 100% ethanol were added to the transferred aqueous phase, and thorough mixing was achieved by pipetting. Then, 700 μl of sample was transferred to the column and centrifuged at 13,000 rpm for 15 sec at room temperature, and the flow-through was discarded. Then, 700 μl of Buffer RWT was added to each column and centrifuged again at 13,000rpm for 15sec at room temperature, and the flow-through was discarded. Then, 500μl Buffer RPE was added to the column and centrifuged at 13,000rpm for 15sec at room temperature, and the flow-through was discarded. Then, another 500μl Buffer RPE was added to the column and centrifuged at 13,000rpm for 2min at room temperature, and the flow-through was discarded. Then, the column was placed in a new 2ml collection tube and centrifuged at 13,000rpm for 1min at room temperature to dry it. The column was transferred to a new 1.5ml collection tube. To elute total RNA (including microRNA), 40μl RNase-free water was drawn onto the column and incubated for 1min, and centrifuged at 13.000rpm for 1min at room temperature. Then, the eluate was placed back into the same column, incubated at room temperature for 1min and centrifuged again for 1min. Using NanoDrop The eluted total RNA (including microRNA) was quantified at 1000 μg and stored at -20°C before use in expression profiling experiments. For quality control of total RNA, 1 μl of total RNA was applied to Agilent's Bioanalyzer and either Agilent's nano- or pico-RNA Chip was selected depending on the RNA concentration determined by NanoDrop measurement.

Example 3: Determination of expression profiles based on microarrays

Using Geniom Biochip miRNA Homo sapiens, microarray hybridization was used to analyze total RNA samples including miRNA fractions (obtained by the protocol of Example 2) on a Geniom real-time analyzer (febitbiomed GmbH, Heidelberg, Germany). Each microfluidic microarray contained complementary DNA probes for 866 miRNAs and miRNA* annotated in Sanger miRBase 12.0 (each represented by 7 repeats). Samples labeled with biotin were analyzed using febit's MPEA analysis by enzymatic on-chip labeling of miRNAs. After hybridization at 42°C for 16 hours, the biochip was automatically washed and processed for signal enhancement using the Geniom real-time analyzer. The resulting detection images were evaluated using Geniom Wizard software. For each array, the median signal intensity was extracted from the raw data file so that for each miRNA, seven intensity values corresponding to each repeated copy of miRBase on the array had been calculated. After background correction, the seven repeated intensity values for each miRNA were summarized by their median values. To standardize the data between different arrays, quantile normalization was applied and all further analyses were performed using normalized and background-subtracted intensity values. From median g1 and median g2, the fold change in expression (=qmedian) was calculated as the ratio g1/g2.

Example 4: Statistical Analysis

After having verified the normal distribution of the measurement data, a parametric T test (unpaired, two-tailed) was performed on each miRNA to detect miRNAs that showed different behaviors in different blood donor groups. The original p value obtained was corrected for multiple tests by Benjamini-Hochberg correction (=ttest_adj). In addition, we applied the limma test to each miRNA and corrected it according to Benjamini-Hochberg (=limma_adj). In addition, we applied the receiver operating characteristic and calculated the "area under the curve" value (=AUC). T test value, limma-test value and AUC value allow to judge the statistical significance of the differential expression of each miRNA between group 1 (g1 subject) and group 2 (g2 subject).

Example 5: Determination of disease-regulated miRNAs and disease-maintaining miRNAs

The goal of this study is to identify miRNAs that show significantly changed expression in different diseases and healthy controls, as well as miRNAs that show very low expression variability in diseases and controls. These two groups of miRNAs are hereinafter referred to as "disease-regulated" miRNAs or "disease-maintained" miRNAs. A total of 1,049 blood samples were obtained for miRNA profiles, all of which were collected in PAXgene blood RNA tubes using the same protocol. In each case, a spectrogram was generated by the expression values of 848 kinds of miRNAs recorded by microarray analysis. To identify "disease-regulated" miRNAs or "disease-maintained" miRNAs, we used T tests and AUC values. Using 20 different thresholds for p values or AUC values, we obtained different groups of "disease-regulated" miRNAs (Fig. 1) or "disease-maintained" miRNAs (Fig. 2).

Sequence Listing

<110> HUMMINGBIRD DIAGNOSTICS GMBH

<120> Health Sign

<130> 17C82562CN

<160> 77

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> RNA

<213> Homo sapiens

<400> 1

ugaggggcag agagcgagac uuu 23

<210> 2

<211> 21

<212> RNA

<213> Homo sapiens

<400> 2

uucaaguaau ucaggauagg u 21

<210> 3

<211> 22

<212> RNA

<213> Homo sapiens

<400> 3

uuauaauaca accugauaag ug 22

<210> 4

<211> 23

<212> RNA

<213> Homo sapiens

<400> 4

guccaguuuu cccaggaauc ccu 23

<210> 5

<211> 17

<212> RNA

<213> Homo sapiens

<400> 5

ucucgcuggg gccucca 17

<210> 6

<211> 23

<212> RNA

<213> Homo sapiens

<400> 6

caaagugcuc auagugcagg uag 23

<210> 7

<211> 22

<212> RNA

<213> Homo sapiens

<400> 7

ggauaucauc auauacugua ag 22

<210> 8

<211> 23

<212> RNA

<213> Homo sapiens

<400> 8

caaagugcuu acagugcagg uag 23

<210> 9

<211> 22

<212> RNA

<213> Homo sapiens

<400> 9

ucguaccgug aguaauaaug cg 22

<210> 10

<211> 23

<212> RNA

<213> Homo sapiens

<400> 10

uaaagugcuu auagugcagg uag 23

<210> 11

<211> 22

<212> RNA

<213> Homo sapiens

<400> 11

auauaauaca accugcuaag ug 22

<210> 12

<211> 23

<212> RNA

<213> Homo sapiens

<400> 12

uaaggugcau cuagugcaga uag 23

<210> 13

<211> 23

<212> RNA

<213> Homo sapiens

<400> 13

aaaagugcuu acagugcagg uag 23

<210> 14

<211> 21

<212> RNA

<213> Homo sapiens

<400> 14

uaaagugcug acagugcaga u 21

<210> 15

<211> 21

<212> RNA

<213> Homo sapiens

<400> 15

agcuacaucu ggcuacuggg u 21

<210> 16

<211> 22

<212> RNA

<213> Homo sapiens

<400> 16

ugccuacuga gcugaaacac ag 22

<210> 17

<211> 22

<212> RNA

<213> Homo sapiens

<400> 17

uucaaguaau ccaggauagg cu 22

<210> 18

<211> 23

<212> RNA

<213> Homo sapiens

<400> 18

caaagugcug uucgugcagg uag 23

<210> 19

<211> 23

<212> RNA

<213> Homo sapiens

<400> 19

ugugcaaauc caugcaaaac uga 23

<210> 20

<211> 22

<212> RNA

<213> Homo sapiens

<400> 20

ugucaguuug ucaaauaccc ca 22

<210> 21

<211> 22

<212> RNA

<213> Homo sapiens

<400> 21

acugcugagc uagcacuucc cg 22

<210> 22

<211> 21

<212> RNA

<213> Homo sapiens

<400> 22

gugggcgggg gcaggugugu g 21

<210> 23

<211> 22

<212> RNA

<213> Homo sapiens

<400> 23

ucaggcucag uccccucccg au 22

<210> 24

<211> 22

<212> RNA

<213> Homo sapiens

<400> 24

aauugcacgg uauccaucug ua 22

<210> 25

<211> 22

<212> RNA

<213> Homo sapiens

<400> 25

ucagugcacu acagaacuuu gu 22

<210> 26

<211> 22

<212> RNA

<213> Homo sapiens

<400> 26

gagcuuauuc auaaaagugc ag 22

<210> 27

<211> 22

<212> RNA

<213> Homo sapiens

<400> 27

uauugcacuu gucccggccu gu 22

<210> 28

<211> 22

<212> RNA

<213> Homo sapiens

<400> 28

uagcagcacg uaaauauugg cg 22

<210> 29

<211> 23

<212> RNA

<213> Homo sapiens

<400> 29

ugugcaaauc uaugcaaaac uga 23

<210> 30

<211> 22

<212> RNA

<213> Homo sapiens

<400> 30

caggccauau ugugcugccu ca 22

<210> 31

<211> 22

<212> RNA

<213> Homo sapiens

<400> 31

cugguacagg ccugggggac ag 22

<210> 32

<211> 21

<212> RNA

<213> Homo sapiens

<400> 32

caacaccagu cgaugggcug u 21

<210> 33

<211> 22

<212> RNA

<213> Homo sapiens

<400> 33

aguuuugcau aguugcacua ca 22

<210> 34

<211> 21

<212> RNA

<213> Homo sapiens

<400> 34

uucacagugg cuaaguucug c 21

<210> 35

<211> 21

<212> RNA

<213> Homo sapiens

<400> 35

uaccacaggg uagaaccacg g 21

<210> 36

<211> 23

<212> RNA

<213> Homo sapiens

<400> 36

aaugacacga ucacucccgu uga 23

<210> 37

<211> 22

<212> RNA

<213> Homo sapiens

<400> 37

uguaaacauc cuacacucag cu 22

<210> 38

<211> 22

<212> RNA

<213> Homo sapiens

<400> 38

aagcugccag uugaagaacu gu 22

<210> 39

<211> 22

<212> RNA

<213> Homo sapiens

<400> 39

cauugcacuu gucucggucu ga 22

<210> 40

<211> 22

<212> RNA

<213> Homo sapiens

<400> 40

uguaacagca acuccaugug ga 22

<210> 41

<211> 21

<212> RNA

<213> Homo sapiens

<400> 41

cugaccuaug aauugacagc c 21

<210> 42

<211> 23

<212> RNA

<213> Homo sapiens

<400> 42

ucucacacag aaaucgcacc cgu 23

<210> 43

<211> 22

<212> RNA

<213> Homo sapiens

<400> 43

uagcagcaca uaaugguuug ug 22

<210> 44

<211> 19

<212> RNA

<213> Homo sapiens

<400> 44

aaaagcuggg uugagagga 19

<210> 45

<211> 20

<212> RNA

<213> Homo sapiens

<400> 45

aaaagcuggg uugagagggu 20

<210> 46

<211> 20

<212> RNA

<213> Homo sapiens

<400> 46

acugccccag gugcugcugg 20

<210> 47

<211> 22

<212> RNA

<213> Homo sapiens

<400> 47

ugagguagua gauuguauag uu 22

<210> 48

<211> 22

<212> RNA

<213> Homo sapiens

<400> 48

ucggccugac cacccacccc ac 22

<210> 49

<211> 22

<212> RNA

<213> Homo sapiens

<400> 49

aaggagcuca cagucuauug ag 22

<210> 50

<211> 23

<212> RNA

<213> Homo sapiens

<400> 50

cgcauccccu agggcauugg ugu 23

<210> 51

<211> 22

<212> RNA

<213> Homo sapiens

<400> 51

ucugggcaac aaagugagac cu 22

<210> 52

<211> 22

<212> RNA

<213> Homo sapiens

<400> 52

cagcagcaau ucauguuuug aa 22

<210> 53

<211> 21

<212> RNA

<213> Homo sapiens

<400> 53

ugagaugaag cacuguagcu c 21

<210> 54

<211> 22

<212> RNA

<213> Homo sapiens

<400> 54

cacccguaga accgaccuug cg 22

<210> 55

<211> 23

<212> RNA

<213> Homo sapiens

<400> 55

ucccccaggu gugauucuga uuu 23

<210> 56

<211> 21

<212> RNA

<213> Homo sapiens

<400> 56

gcccuccgcc cgugcacccc g 21

<210> 57

<211> 22

<212> RNA

<213> Homo sapiens

<400> 57

ugugcgcagg gagaccucuc cc 22

<210> 58

<211> 20

<212> RNA

<213> Homo sapiens

<400> 58

cugcaaaggg aagcccuuuc 20

<210> 59

<211> 22

<212> RNA

<213> Homo sapiens

<400> 59

cugcccuggc ccgagggacc ga 22

<210> 60

<211> 23

<212> RNA

<213> Homo sapiens

<400> 60

agcagcauug uacagggcua uca 23

<210> 61

<211> 21

<212> RNA

<213> Homo sapiens

<400> 61

uggcagggag gcugggaggg g 21

<210> 62

<211> 18

<212> RNA

<213> Homo sapiens

<400> 62

cgggcguggu gguggggg 18

<210> 63

<211> 23

<212> RNA

<213> Homo sapiens

<400> 63

agcagcauug uacagggcua uga 23

<210> 64

<211> 20

<212> RNA

<213> Homo sapiens

<400> 64

uacaguauag augauguacu 20

<210> 65

<211> 22

<212> RNA

<213> Homo sapiens

<400> 65

uucaccaccu ucuccaccca gc 22

<210> 66

<211> 18

<212> RNA

<213> Homo sapiens

<400> 66

aucccaccuc ugccacca 18

<210> 67

<211> 23

<212> RNA

<213> Homo sapiens

<400> 67

agcucggucu gaggccccuc agu 23

<210> 68

<211> 23

<212> RNA

<213> Homo sapiens

<400> 68

uagugcaaua uugcuuauag ggu 23

<210> 69

<211> 22

<212> RNA

<213> Homo sapiens

<400> 69

ucagcaaaca uuuauugugu gc 22

<210> 70

<211> 22

<212> RNA

<213> Homo sapiens

<400> 70

caaaccacac ugugguguua ga 22

<210> 71

<211> 21

<212> RNA

<213> Homo sapiens

<400> 71

gugcauugua guugcauugc a 21

<210> 72

<211> 22

<212> RNA

<213> Homo sapiens

<400> 72

cuauacgacc ugcugccuuu cu 22

<210> 73

<211> 22

<212> RNA

<213> Homo sapiens

<400> 73

aaaagcuggg uugagagggc ga 22

<210> 74

<211> 22

<212> RNA

<213> Homo sapiens

<400> 74

aaugcaccug ggcaaggauu ca 22

<210> 75

<211> 22

<212> RNA

<213> Homo sapiens

<400> 75

caacaaauca cagucugcca ua 22

<210> 76

<211> 23

<212> RNA

<213> Homo sapiens

<400> 76

cucucaccac ugcccuccca cag 23

<210> 77

<211> 21

<212> RNA

<213> Homo sapiens

<400> 77

cccggagcca ggaugcagcu c 21

Claims (4)

1. Use in the preparation of diagnostic solutions/devices for the in vitro assessment of the health status of subjects in total RNA of blood cells from whole blood samples taken from subjects, for the determination of at least two polynucleotide groups of the following miRNAs: (ia) One or more miRNAs included in a first predetermined group of miRNAs, wherein the miRNAs are selected from SEQ ID NO:1 to SEQ ID NO:16 and SEQ ID NO:20 to SEQ ID NO:26, and (ib) One or more miRNAs contained in a second predetermined group of miRNAs, wherein the miRNAs are selected from SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43 to SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:55 to SEQ ID NO:59. 2. The use as described in claim 1, wherein the diagnostic device is a diagnostic kit. 3. Use in the preparation of diagnostic solutions/devices for the in vitro assessment of the following polynucleotide groups of at least two miRNAs in total RNA from blood cells in whole blood samples taken from subjects, for the determination of miRNAs: (ia) One or more miRNAs included in a first predetermined group of miRNAs, wherein the miRNAs are selected from SEQ ID NO:1 to SEQ ID NO:16 and SEQ ID NO:20 to SEQ ID NO:26, and (ib) One or more miRNAs contained in a second predetermined group of miRNAs, wherein the miRNAs are selected from SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43 to SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:55 to SEQ ID NO:59. 4. The use as described in claim 3, wherein the diagnostic device is a diagnostic kit.