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WO2017032815A2 - Biomarqueurs pour maladies cardiométaboliques - Google Patents

Biomarqueurs pour maladies cardiométaboliques Download PDF

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Publication number
WO2017032815A2
WO2017032815A2 PCT/EP2016/070011 EP2016070011W WO2017032815A2 WO 2017032815 A2 WO2017032815 A2 WO 2017032815A2 EP 2016070011 W EP2016070011 W EP 2016070011W WO 2017032815 A2 WO2017032815 A2 WO 2017032815A2
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Prior art keywords
protein
amount
masp
mrna
proteins
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WO2017032815A3 (fr
Inventor
Christine VON TÖRNE
Stefanie M. HAUCK
Cornelia HUTH
Barbara THORAND
Tonia DE LAS HERAS GALA
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Publication of WO2017032815A3 publication Critical patent/WO2017032815A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to a method of identifying a predisposition for developing type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a type 2 diabetes mellitus, said method comprising or consisting of (i) assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from a group of proteins as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of APOC2, APOC3, APOE, CRP, MASP-1 , ADIPOQ, ITIH4, and SHBG; (b) said parameters are age, sex and BMI, and said group consists of APOC2, APOC
  • Cardiometabolic diseases include diseases such as type 2 diabetes mellitus, hypertension, metabolic syndrome, renal failure and heart failure and are among the most severe health problems worldwide.
  • Cardiometabolic risk represents the overall risk of developing cardiovascular diseases and type 2 diabetes, caused by the clustering of modifiable risk factors. These include classical cardiovascular risk factors such as smoking, high LDL cholesterol, hypertension, elevated blood glucose and emerging risk factors closely related to abdominal obesity, such as insulin resistance, low HDL, high triglycerides and inflammatory markers. This clustering of risk factors is often operationalized by the definition of the metabolic syndrome.
  • cardiometabolic diseases are considered to have genetic and environmental etiologies (Lopez-Jaramillo et al., Ther Adv Cardiovasc Dis. 2011 Apr;5(2):119-31) and can be present long before becoming clinically apparent. As such, there is a need not only to provide adequate treatment options but also to provide methods for early recognition, i.e. diagnosis and prognosis, of these cardiometabolic diseases.
  • type 2 diabetes mellitus is a metabolic disease defined by increased blood glucose levels due to beta-cell dysfunction and insulin resistance which develops slowly over a long time span, without evidence for specific causes such as autoimmune destruction of beta-cells (Stumvoll et al., Lancet 365, 1333-1346 (2005); Muoio et al., Nat Rev Mol Cell Biol 9, 193-205 (2008)).
  • a state of prediabetes with only slightly higher blood glucose levels may precede the disease for years (McGarry et al., Diabetes 51 , 7-18 (2002)) and increase also the risk for cardiovascular diseases (Schwarz et al., Horm Metab Res 41 , 86-97 (2009)).
  • T2D pathophysiological processes
  • the development of T2D in prediabetic individuals can be prevented or delayed by dietary changes and increased physical activity (Tuomilehto et al., N Engl J Med 344, 1343-1350 (2001 )). Risk prediction for T2D remains suboptimai even after the introduction of global risk assessment by various scores.
  • the known risk factors for T2D include age, sex, anthropometric (obesity.height), metabolic (blood pressure, blood lipids, liver enzymes, uric acid, leukocyte count, C-reactive protein and adiponectin), socioeconomic (education, income) and life style (physical inactivity, dietary components, smoking and alcohol intake) variables (Lloyd et al., Int J Obes (Lond) 2012, 36(1 ):1 -11 ; Gerich JE, Clinical cornerstone 2007, 8(3):53-68; Grundy SM, J Am Coll Cardiol 2012, 59(7):635-643; Aschner P, Expert review of cardiovascular therapy 2010, 8(3):407-412; Lechleitner M, Gerontology 2008, 54(5):253-259; Buijsse B et al., Epidemiol Rev 2011 ;33:46-62).
  • T2D Therapeutic therapy
  • Life style intervention e.g. dietary modification, exercise training and weight loss
  • Improvement of risk prediction for T2D is crucial to the identification of high-risk individuals who could benefit from targeted preventive measures.
  • the technical problem underlying the present invention was to identify alternative and/or improved means and methods to identify subjects who are at risk to develop a cardiometabolic disease, such as, e.g., T2D.
  • a cardiometabolic disease such as, e.g., T2D.
  • the solution to this technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention provides embodiments on the one hand which relate to diagnosing predispositions for certain diseases.
  • Said diseases are generally in the field of cardiometabolic diseases.
  • Preferred cardiometabolic diseases are T2D and prediabetes.
  • a distinction is made between identifying a predisposition for T2D (without considering prediabetes) and a predisposition for prediabetes and/or T2D.
  • the present invention relates to a method of identifying a predisposition for developing a cardiometabolic disease in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease, said method comprising of consisting of the step of assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from mannan-binding lectin serine peptidase 1 (MASP-1 ), glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1 ), thrombospondin (THBS1) and apolipoprotein A-IV (APOA-IV), wherein an increase in the amount of MASP-1 , GPLD1 , or THBS
  • MASP-1
  • predisposition for developing a disease is established in the art and used herein analogously. Therefore, the term “predisposition for developing a cardiometabolic disease” describes the status of a patient at risk for developing a cardiometabolic disease. As used herein, the predisposition to develop a cardiometabolic disease is based on environmental, genetic and/or epigenetic factors.
  • a "subject' in accordance with the method of the invention is preferably a human subject.
  • the human subject can be a patient, i.e. be subjected to supervision or treatment by medical staff.
  • the invention relates in an alternative embodiment to a method for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease.
  • the one or more marker(s) i.e. the one or more protein(s) and/or mRNA(s) as specified, are used to enhance the diagnostic/predictive value of an existing diagnosis/prediction with respect to a cardiometabolic disease.
  • the diagnostically informative values are used to aid in identifying said predisposition, wherein said values are combined with existing diagnostic/prognostic values from different diagnostic and/or prognostic methods to allow a prediction of the development of a cardiometabolic disease.
  • Said diagnostically informative value is based on the determination according to the claimed method steps that the protein level or mRNA level of said one or more protein(s) is disturbed in comparison to control levels in the direction specified herein above.
  • Said one or more proteins are those specified in the first embodiment, i.e. MASP-1 , GPLD1 , THBS1 and APOA-IV.
  • the present invention furthermore provides the use of one, two, three, four, five, six, seven, eight, nine or ten marker proteins selected from ADIPOQ, APOA-IV, APOC2, APOC3, APOE, C7, CD14, CRP, ITIH4, MASP1 , ORM1 , PZP, SHBG, GPLD1 , LBP and RBP4 for identifying a predisposition for developing type 2 diabetes mellitus, wherein a significantly increased protein or mRNA level of APOA-IV, APOC2, APOC3, APOE, C7, CD14, CRP, MASP1 and LBP and a significantly decreased protein or mRNA level of ADIPOQ, ITIH4, ORM1 , PZP, SHBG, GPLD1 and RBP4 are indicative of said predisposition.
  • the present invention provides the use of one, two, three, four, five, six, seven, eight, nine or ten marker proteins selected from ADIPOQ, APOA-IV, APOC2, APOC3, APOE, C7, CD14, CRP, ITIH4, MASP1 , ORM1 , PZP, SHBG, GPLD1 , LBP and RBP4 for identifying a predisposition for developing type 2 diabetes mellitus or prediabetes, wherein a significantly increased protein or mRNA level of APOA-IV, APOC2, APOC3, APOE, C7, CD14, CRP, MASP1 and LBP and a significantly decreased protein or mRNA level of ADIPOQ, ITIH4, ORM1 , PZP, SHBG, GPLD1 and RBP4 are indicative of said predisposition.
  • Preferred is the use of protein levels.
  • patients or individuals may be further characterized in the sense that one or more parameters characterizing a given patient or individual are known.
  • parameters include age, sex, body mass index (BMI), smoking status, alcohol intake, physical inactivity, actual hypertension, log-transformed triacylglycerol levels and/or log-transformed total cholesterol/HDL-cholesterol ratio.
  • BMI body mass index
  • smoking status smoking status
  • alcohol intake physical inactivity
  • actual hypertension log-transformed triacylglycerol levels
  • log-transformed total cholesterol/HDL-cholesterol ratio log-transformed total cholesterol/HDL-cholesterol ratio
  • Adjustment means that these further parameters are included in the statistical regression model or statistical prediction or diagnosis algorithm as covariates.
  • said parameters are used as covariates in said methods of identifying a predisposition of the invention.
  • a probability P for predisposition is defined as follows:
  • P(Y 1
  • the present invention also provides a method of identifying a predisposition for developing type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a type 2 diabetes mellitus, said method comprising or consisting of (i) assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from a group of proteins as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and
  • Persons with a cardiometabolic disease as well as persons with a predisposition for a cardiometabolic disease are on average older than persons without a cardiometabolic disease or without a predisposition for a cardiometabolic disease, a preferred cardiometabolic disease being type 2 diabetes mellitus. Moreover, they have a higher BMI on average, are less physically active, are more likely to be male, to smoke or to have smoked, to consume larger amounts of alcohol and to suffer from actual hypertension. Furthermore, they have a higher total cholesterol/HDL-cholesterol ratio, as well as higher triacylglycerol and hsCRP levels.
  • said group consists of (a) APOC2, APOC3, APOE, CRP, MASP-1 , ADIPOQ, ITIH4 and SHBG.
  • said group consists of (b) APOC2, APOC3, APOE, MASP-1 , ADIPOQ, ITIH4, PZP and SHBG.
  • said group consists of (c) MASP-1 , ADIPOQ, ITIH4, ORM1 and PZP.
  • This embodiment extends to implementations where only one marker or a subset of all markers recited in a given item (a), (b) or (c) are used. Obviously, it also extends to implementations where all listed markers are used. Preferably two, three, four, five, six, seven, eight, nine or ten markers are used. This applies to all embodiments reciting "one or more protein(s)".
  • the present invention also provides a method of identifying a predisposition for developing a type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amounts of all members of a group of proteins or mRNAs encoding said proteins for identifying a predisposition for developing a type 2 diabetes mellitus, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amounts of said proteins and/or mRNAs encoding said proteins, wherein said group of proteins is as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of APOE, C7, CRP, MASP-1 , ADIPOQ, ITIH4, ORM1 and SHBG; (b) said parameters are age, sex and BMI, and said group consists of APOE, C7, CD14, MASP-1 , ADIPOQ, ITIH4, ORM1 and
  • the present invention not only provides methods of identifying a predisposition for developing type 2 diabetes, but also methods for identifying predispositions for developing type 2 diabetes mellitus and/or prediabetes.
  • the art recognizes two slightly distinct definitions of prediabetes, one according to ADA, the other according to WHO. This has been taken into account when defining the following embodiments.
  • the following two embodiments employ, for the purpose of defining prediabetes, the definition of ADA.
  • parameters including clinical parameters characterizing patients and/or individuals may be considered when assessing predispositions. This is reflected in items (a), (b) and (c) of the following four embodiments which items (a), (b) and (c) correspond to models 1 , 2 and 3 according to the present invention.
  • the present invention furthermore provides a method of identifying a predisposition for developing prediabetes or type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing prediabetes or type 2 diabetes mellitus, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from a group of proteins as defined in the following, and (ii) determining parameters of said subject, wherein said parameters are age and sex, and said group consists of CRP, MASP1 and SHBG; wherein an increase in the amount of CRP or MASP1 or of an mRNA encoding CRP or MASP1 or a decrease in the amount of SHBG or
  • the present invention also provides a method of identifying a predisposition for developing prediabetes or type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amounts of all members of a group of proteins or mRNAs encoding said proteins for identifying a predisposition for developing prediabetes or type 2 diabetes mellitus, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amounts of said proteins and/or mRNAs encoding said proteins, wherein said group of proteins is as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of C7, CRP, MASP-1 , RBP4 and SHBG; (b) said parameters are age, sex and BMI, and said group consists of MASP-1 and GPLD1 ; or (c) said parameters are age, sex, BMI, smoking status, alcohol intake, physical in
  • the latter two embodiments employ, for the purpose of defining prediabetes, the definition of ADA.
  • the following two embodiments are fully analogous to the preceding two embodiments, but employ the definition of prediabetes according to WHO.
  • the present invention furthermore provides a method of identifying a predisposition for developing prediabetes or type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing prediabetes or type 2 diabetes mellitus, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from a group of proteins as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of C7, CRP, LBP, MASP-1 and SHBG; (b) said parameters are age, sex and BMI, and said group consists of C7 and MASP-1 ; or (
  • the present invention also provides a method of identifying a predisposition for developing prediabetes or type 2 diabetes mellitus in a subject, or for producing a diagnostically informative value of the amounts of all members of a group of proteins or mRNAs encoding said proteins for identifying a predisposition for developing prediabetes or type 2 diabetes mellitus, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amounts of said proteins and/or mRNAs encoding said proteins, wherein said group of proteins is as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of C7, CRP, MASP-1 , SHBG and RBP4; (b) said parameters are age, sex and BMI, and said group consists of C7, LBP and MASP-1; or (c) said parameters are age, sex, BMI, smoking status, alcohol intake, physical in
  • Cardiometabolic disease is known in the art and described, e.g., in Lopez- Jaramillo et al., Ther Adv Cardiovasc Dis. 201 Apr;5(2): 119-31 and used herein accordingly.
  • Cardiometabolic diseases in accordance with the invention include type 2 diabetes mellitus, prediabetes, hypertension, metabolic syndrome, acute coronary syndrome and heart failure. Type 2 diabetes mellitus is preferred.
  • sample refers to a biological sample, such as, for example, cells, tissues (from any organ), or fluids (including serum, plasma, whole blood), which has been isolated or obtained from an individual or from cell culture constituents of a cell culture comprising a subject's cells.
  • tissue or liquid sample obtained from a patient and/or subject can be used for the assessment of the amount of the one or more protein(s) or mRNAs according to the method of the invention.
  • cell types are hepatocytes, cardiomyocytes, adipocytes, myocytes, epithelial cells (of kidney, lung or cardiovascular origin) and fibroblasts, and cells derived from these (e.g. immortalized cell lines or induced pluripotent stem cell lines).
  • RNA/Protein kit Macherey-Nagel
  • the sample may need to be manipulated depending on the type of sample and the method chosen for assessment.
  • blood can be drawn into suitable containers (e.g., tubes such as S-Monovette® serum tubes (SARSTEDT AG & Co., Numbrecht, Germany)) followed by one or more gentle inversions of the containers, and letting the samples rest for 30 minutes at room temperature to obtain complete coagulation.
  • suitable containers e.g., tubes such as S-Monovette® serum tubes (SARSTEDT AG & Co., Numbrecht, Germany)
  • centrifugation of blood e.g. at 2750 g and 15 °C for 10 minutes, can be performed.
  • Serum can then be separated and, if desired, filled into containers, e.g., synthetic straws, for storage, such as in liquid nitrogen (-196 °C), until the execution of protein or mRNA analyses.
  • containers e.g., synthetic straws
  • EDTA plasma standard operating procedures can be used (see e.g., Tuck et al., J Proteome Res. 2009 Jan; 8(1 ): 1 13-117). If analyzing protein, LoBi ' nd tubes (Eppendorf, Hamburg, Germany) are preferred.
  • protein is used in line with its well-known meaning in the art. Briefly, a protein (also referred to herein as “polypeptide”) as used herein describes a group of molecules consisting of more than 30 amino acids, whereas the term “peptide” as used herein describes a group of molecules consisting of up to 30 amino acids.
  • mRNA (which can also be referred to herein as “mRNA molecule”; the abbreviation stands for “messenger RNA”) is well-established in the art and used herein accordingly.
  • mRNA describes a large family of RNA molecules that convey genetic information from DNA to the ribosome, where these molecules are used as matrices for determining the amino acid sequence of products of gene expression, i.e. proteins.
  • the mRNA molecules according to the invention encode the amino acid sequence of the one or more protein(s) identified herein as marker(s). As such, these mRNAs have been transcribed from the genes encoding the respective proteins and are used for translation into said proteins.
  • MASP-1 The protein "MASP-1" (or MASP1 ) is known in the art and described, e.g., in the respective entry in the genecards human database of the Weizmann Institute of Science, Israel (Safran, M. et al, GeneCards Version 3: the human gene integrator Database 2010; doi: 10. 1093/database/baq020).
  • MASP-1 is the most abundant serine protease of the complement lectin pathway.
  • the complement cascade is initiated when a complex of mannose binding lectin (MBL), MBL-associated serine proteases (MASPs: MASP1 , MASP2, MASP3) and the MBP-associated proteins (MAp19 and Map44) binds to its target (Thiel, S.
  • MBL mannose binding lectin
  • MASPs MBL-associated serine proteases
  • MAp19 and Map44 MBP-associated proteins
  • GPLD1 The protein "GPLD1" is known in the art and described, e.g., in Hoener, M. C, and Brodbeck, U. (1992) (Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins. European journal of biochemistry / FEBS 206, 747-757). GPLD1 is an amphiphilic protein, associated with high- density lipoproteins in serum.
  • the protein (SEQ ID NO: 3) and cDNA sequence (SEQ ID NO: 4) of GPLD1 are known in the art and can be found in the Ensembl Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000230036 and ENST00000230036, respectively.
  • THBS1 The protein "THBS1" is known in the art and described, e.g., in the respective entry in the genecards human database of the Weizmann Institute of Science, Israel (loc. cit). THBS1 is an adipokine that is increased in T2D.
  • the protein (SEQ ID NO: 5) and cDNA sequence (SEQ ID NO: 6) of THBS1 are known in the art and can be found in the Ensembl Human database (release 72 (GRCm38.p1)) under accession number ENSP00000260356 and ENST00000260356, respectively.
  • the protein APOA-IV (or APOA4) is known in the art and described, e.g., in the respective entry in the genecards human database of the Weizmann Institute of Science, Israel (loc. cit.), APOA-IV is involved in lipid transport in the blood and occurs in plasma as free APOA- IV or as part of chylomicrons (Green, P. H. et al. (1980) Human apolipoprotein A-IV. Intestinal origin and distribution in plasma. The Journal of clinical investigation 65, 911-919; Bisgaier, C. L. et al. (1985) Distribution of apolipoprotein A-IV in human plasma. Journal of lipid research 26, 11-25).
  • the protein (SEQ ID NO: 7) and cDNA sequence (SEQ ID NO: 8) of APOA-IV are known in the art and can be found in the Ensembl Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000350425 and ENST00000357780, respectively.
  • the primary translation product is a 396-residue preprotein which after proteolytic processing is secreted at its primary site of synthesis, the intestine, in association with chylomicron particles.
  • assessing the amount refers to any method that can be employed to quantify the presence of one or more protein(s) or mRNA(s).
  • the person skilled in the art is aware of experimental protocols that are suitable to determine the amount of one or more protein(s) or mRNA(s). For example, methods such mass spectrometry alone or in combination with, e.g., gas/liquid chromatography can be employed to assess the amount of said one or more protein(s) in a sample.
  • Further methods to quantify the amount of said one or more protein(s) are known in the art and include, e.g., the enzyme-linked immunosorbent assay (ELISA), Western blot analysis.
  • the quantification is an absolute quantification.
  • the assessment of the "amount" of said one or more protein(s) or mRNA(s) is the decisive factor in the process of diagnosing a risk to develop a cardiometabolic disease in accordance with the method of the invention.
  • the amounts of proteins or mRNAs generally and normally vary from subject to subject depending on age, sex and/or condition, inter alia, to a certain extent. As such, the amounts can be normalized.
  • Several strategies for the normalisation of protein and mRNA concentrations are known in the art, including, without being limiting, normalising against the concentration of an internal reference, which is determined in the same sample, normalisation against sample size, normalization against total protein or mRNA amount or normalisation against an artificially introduced molecule of known amount.
  • normalisation may also be carried out by adjusting the obtained values by patient-specific factors such as e.g. age, BMI, hormone status, nutritional factors (e.g. fasting) or time (circadian rhythm). Normalization can, for example, be achieved by dividing the measured values of the protein(s) or mRNA(s) to be investigated by the measured values of a reference molecule or by subtracting the measured values of the reference molecule from the measured value of the protein(s) or mRNA(s) of interest. Variations to be considered not normal, e.g., variations in protein or mRNA amounts linked to a disease, have to be, preferably statistically, significant when compared to a suitable control population.
  • patient-specific factors such as e.g. age, BMI, hormone status, nutritional factors (e.g. fasting) or time (circadian rhythm).
  • Normalization can, for example, be achieved by dividing the measured values of the protein(s) or mRNA(s) to be investigated by the measured values of a reference molecule
  • Mass spectrometry and its use for determining the amount/concentration of proteins in a sample is well known in the art and has been described, for example in Liebler and Zimmerman (Biochemistry. 2013 Jun 4;52(22):3797-806) and the Nature Methods Special Issue "SRM-Method of the year” (Nature Methods, Vol. 10 No1 , January 2013).
  • von Toerne et al. J Proteome Res. 2013 Mar 1 ;12(3): 1331 -43 have described an optimized rapid workflow for SRM-based quantification of protein levels to assess various proteins in NZO mice with a diabetic phenotype.
  • mass spectrometry involves ionizing a molecule and then measuring the mass of the resulting ions. Since molecules ionize in a way that is well known, the molecular weight of the molecule can be accurately determined from the mass of the ions. In addition, by a comparison of data obtained from internal standards, a quantification of molecules of interest is possible, as detailed herein below.
  • Mass spectrometry methods that can be employed in accordance with the invention and which allow assessing the amount of one or more protein(s) include, for example, tandem mass spectrometry and non-targeted (Shotgun) and targeted mass spectrometry. Preferably, targeted mass spectrometry is used.
  • Tandem mass spectrometry involves first obtaining a mass spectrum of the ion of interest, then fragmenting that ion and obtaining a mass spectrum of the fragments. Tandem mass spectrometry thus provides both molecular weight information and a fragmentation pattern that can be used in combination along with the molecular weight information to identify the exact sequence of a peptide or protein (see e.g. Hunt et al. (1986) PNAS USA 83:6233- 6237; Shevchenko et al. (1996) PNAS USA 93:14440-14445; Figeys et al. (1996) Anal. Chem. 68:1822-1828 and Wilm et al.
  • Targeted mass spectrometry is a mass spectrometry technique relying on the analysis of ions of specific m/z value and is described in detail, for example, in Nature Methods Special Issue "SRM-Method of the year" (Nature Methods, Vol. 10 No1 , January 2013).
  • targeted mass spectrometry such as, e.g., selected reaction monitoring, measures and selects peptide m/z values in a pre-defined, tailored fashion.
  • multiple collision induced dissociation products of peptides are measured in a pre-defined and tailored fashion. Quantification is based on detected dissociation product intensities.
  • MRM multiple reaction monitoring
  • MRM-MS selected reaction monitoring mass spectrometry
  • SRM-MS selected reaction monitoring mass spectrometry
  • protein/peptide amounts are determined by reference to internal protein/peptide standards.
  • Protein quantification methods based on mass spectrometry are known in the art (see, e.g., Pan et al. J Proteome Res. 2009 Feb;8(2):787-9).
  • the abundance of any molecular ion species typically carries information on concentration, but ion abundance is confounded by a number of features, including instrument response factors, ionisation efficiency of the molecule, stability of the molecular ion species and the presence of other molecules that could cause ion suppression of the analyte of interest.
  • internal standards have been developed that can be used to generate appropriate calibration curves to convert abundance of ions into a quantitative measure of protein amounts.
  • Non-limiting examples of internal standards include protein standards labeled with stable isotope-labeled versions of the protein to be quantified, which have a similar extraction recovery, ionization response and a similar chromatographic retention time; compound analogues of the protein to be quantified which are similar to the compound to be quantified but slightly different by parent mass.
  • the internal protein standard is typically added at a known concentration into every sample at the beginning of the sample preparation for mass spectrometric measurement. This way, quality of eventual washing, modification steps and finally the enzymatic digest of proteins into peptides can be monitored. It will be appreciated by the skilled person that the amount of the internal standard needs to be higher than the limit of quantitation but low enough to avoid a suppression of the ionization of the analyte.
  • internal peptide standards preferably isotope labeled, are measured to ensure validity of signal of the sample. These internal peptide standards serve as reference for quantification. Based on the known concentration of the internal standard present in the sample, sample preparation and measurement can be monitored.
  • the measured values for the protein of interest can be quantified by interpolating the response ratio between the protein and the internal peptide standard to a standard curve.
  • the internal peptide standards are stable isotope-labeled standards.
  • stable in the context of isotopes means that the isotopes are not radioactive, i.e. they do not decay spontaneously.
  • Peptide standards labeled with (a) stable isotope(s) are stable isotope-labeled peptides representing the protein to be quantified and are well known in the art.
  • the isotopes employed are stable isotopes of carbon, nitrogen or hydrogen, such as e.g. 12 C and 13 C, 14 N and 5 N and 2 H (Deuterium).
  • the labeled internal peptide standards are the peptide sequences identified herein below in relation to the kit of the invention.
  • Methods for quantification of mRNA molecules are also well-known in the art. Methods include without limitation, e.g., northern blotting, and real-time PCR-based methods (for example RT-PCR), all of which are standard methods in the art.
  • a Northern blot allows the determination of RNA or isolated mRNA in a sample. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. Initially, total RNA extraction from the sample is performed. If desired, mRNA can be separated from said initial RNA sample through the use of oligo (dT) cellulose chromatography to isolate only the RNA with a poly(A) tail.
  • oligo (dT) cellulose chromatography to isolate only the RNA with a poly(A) tail.
  • RNA samples are then separated by gel electrophoresis.
  • the separated RNA is then transferred to a nylon membrane through a capillary or vacuum blotting system.
  • the RNA is immobilized through covalent linkage to the membrane by, e.g., UV light or heat.
  • the RNA is detected using suitably labeled probes and X-ray film and can subsequently be quantified by densitometry.
  • Suitable compositions of gels, buffers and labels are well-known in the art and may vary depending on the specific sample and target to be identified.
  • PCR is well known in the art and is employed to make large numbers of copies of a target sequence. This is done on an automated cycler device, which can heat and cool containers with the reaction mixture in a very short time.
  • the PCR generally, consists of many repetitions of a cycle which consists of: (a) a denaturing step, which melts both strands of a DNA molecule and terminates all previous enzymatic reactions; (b) an annealing step, which is aimed at allowing the primers to anneal specifically to the melted strands of the DNA molecule; and (c) an extension step, which elongates the annealed primers by using the information provided by the template strand.
  • PCR can be performed, for example, in a 50 ⁇ reaction mixture containing 5 ⁇ of 10 x PCR buffer with 1.5 mM MgCI 2 , 200 ⁇ of each deoxynucleoside triphosphate, 0.5 ⁇ of each primer (10 ⁇ ), about 10 to 100ng of template DNA and 1 to 2.5 units of Taq polymerase.
  • the primers for the amplification may be labeled or be unlabeled.
  • DNA amplification can be performed, e.g., with a Applied Biosystems Veriti® Thermal Cycler (Life Technologies Corporation, Carlsbad, CA), C1000TM thermal cycler (Bio-Rad Laboratories, Hercules, CA,), or SureCycler 8800 (Agilent Technologies, Santa Clara, CA): 2 min at 94°C, followed by 30 to 40 cycles consisting of annealing (e. g. 30 s at 50°C), extension (e. g. 1 min at 72°C, depending on the length of DNA template and the enzyme used), denaturing (e. g. 10 s at 94°C) and a final annealing step, e.g.
  • annealing e. g. 30 s at 50°C
  • extension e. g. 1 min at 72°C, depending on the length of DNA template and the enzyme used
  • denaturing e. g. 10 s at 94°C
  • Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus Vent, Amplitaq, Pfu and KOD, some of which may exhibit proof-reading function and/or different temperature optima.
  • E. coli DNA polymerase I or its Klenow fragment T4 DNA polymerase, Tth polymerase, Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus Vent, Amplitaq, Pfu and KOD, some of which may exhibit proof-reading function and/or different temperature optima.
  • it is well known in the art how to optimize PCR conditions for the amplification of specific nucleic acid molecules with primers of different length and/or composition or to scale down or increase the volume of the reaction mix.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • reverse transcriptase refers to an enzyme that catalyzes the polymerization of deoxyribonucleoside triphosphates to form primer extension products that are complementary to a ribonucleic acid template. The enzyme initiates synthesis at the 3' -end of the primer and proceeds toward the 5'-end of the template until synthesis terminates.
  • RNA target sequence into a complementary, copy-DNA (cDNA) sequence examples include avian myeloblastosis virus reverse transcriptase and Thermus thermophilus DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer.
  • cDNA duplex template is heat denatured during the first denaturation step after the initial reverse transcription step leaving the DNA strand available as an amplification template. High-temperature RT provides greater primer specificity and improved efficiency.
  • the RT reaction can be performed, for example, in a 20 ⁇ reaction mix containing: 4 ⁇ of 5x AMV-RT buffer, 2 ⁇ of oligo dT (100 pg/rnl), 2 ⁇ of 10 mM dNTPs, 1 ⁇ total RNA, 10 Units of AMV reverse transcriptase, and H 2 0 to 20 ⁇ final volume.
  • the reaction may be, for example, performed by using the following conditions: The reaction is held at 70°C for 15 minutes to allow for reverse transcription. The reaction temperature is then raised to 95°C for 1 minute to denature the RNA-cDNA duplex.
  • reaction temperature undergoes two cycles of 95°C for 15 seconds and 60°C for 20 seconds followed by 38 cycles of 90°C for 15 seconds and 60°C for 20 seconds. Finally, the reaction temperature is held at 60°C for 4 minutes for the final extension step, cooled to 15°C, and held at that temperature until further processing of the amplified sample.
  • Any of the above mentioned reaction conditions may be scaled up according to the needs of the particular case.
  • the resulting products are loaded onto an agarose gel and band intensities are compared after staining the nucleic acid molecules with an intercalating dye such as ethidium bromide or SybrGreen. A lower band intensity of the sample treated with the inhibitor as compared to a non-treated sample indicates that the inhibitor successfully inhibits the protein.
  • Real-time PCR employs a specific probe, in the art also referred to as TaqMan probe, which has a reporter dye covalently attached at the 5' end and a quencher at the 3' end.
  • TaqMan probe After the TaqMan probe has been hybridized in the annealing step of the PCR reaction to the complementary site of the polynucleotide being amplified, the 5' fluorophore is cleaved by the 5' nuclease activity of Taq polymerase in the extension phase of the PCR reaction. This enhances the fluorescence of the 5' donor, which was formerly quenched due to the close proximity to the 3' acceptor in the TaqMan probe sequence.
  • a control is a subject or group of subjects negative for cardiometabolic diseases, especially type 2 diabetes mellitus.
  • a control is preferably selected from i) a group of subjects (termed herein also "control group") negative for cardiometabolic diseases and is representative (with regard to, e.g., same distribution of age, race, sex, health status) for the investigated population, and/or ii) database entries of corresponding groups of subjects as defined in i).
  • Said group of subjects preferably consist of a suitably large number of subjects, so as to yield representative results.
  • the amounts in samples of at least (for each value) 4, 6, 8, 10, 20, 50, 100, 200 or more preferred at least 400, even more preferred at least (for each value) 500, 600, 700, 800 and most preferred 1000 and more control subjects are determined.
  • control group according to i) does not comprise individuals with protein or mRNA amounts indicative for the person to be at risk of developing a cardiometabolic disease. Nevertheless, a control group according to i) may be normalized to exclude subjects which are at risk to develop a cardiometabolic disease, for example, on the basis of biomarkers other than those according to the invention, so as to (further) refine the initial control data. Conceivably, increasing the number of subjects in the control group will result in a more accurate and representative result to be used as normal amount of a given protein or mRNA in a control. Also preferred is that the protein or mRNA amounts are determined more than once or that several samples of a subject of the control group are assessed.
  • the data of said several samples, said several tests and/or of said subjects of the control group is pooled to calculate the mean or median and, optionally, the variance for each subject in the case of several samples or tests, and/or the variance for the group of control subjects.
  • These values may, e.g., be deposited into a database as a standardized value for each of said one or more protein(s) or mRNA(s) and, if required, be retrieved from a database, hence making the need to also experimentally assess the protein or mRNA amounts in a control sample every time the amount in a patient sample is assessed dispensable.
  • a control may also be a database entry, i.e. the control according to item ii) above.
  • control for the embodiments herein relating to a method of identifying a predisposition for developing a type 2 diabetes mellitus in a subject is more preferably selected from i) a group of subjects (termed herein also "control group") negative for T2D, i.e.
  • control group has a fasting plasma glucose level ⁇ 126 mg/dl and preferably also a two-hour post-glucose load plasma level ⁇ 200 mg/dl during an oral glucose tolerance test with 75 g glucose (in other terms, exhibits no diabetes according to ADA and according to WHO definition), and, optionally displays a HbA1c ⁇ 6.5% and is representative (with regard to, e.g., same distribution of age, race, sex, health status) for the investigated population, and/or ii) database entries of corresponding groups of subjects as defined in i).
  • control for the embodiments herein relating to a method of identifying a predisposition for developing prediabetes or type 2 diabetes mellitus in a subject is more preferably selected from i) a group of subjects (termed herein also "control group") negative for T2D, exhibiting no impaired glucose tolerance (IGT) and, exhibiting no isolated impaired fasting glucose (i-IFG), i.e.
  • control group according to WHO definition has a fasting plasma glucose level ⁇ 110 mg/dl and a two-hour post-glucose load plasma level ⁇ 140 mg/dl during an oral glucose tolerance test with 75 g glucose (in other terms, exhibits no prediabetes according to WHO definition);
  • control group according to ADA definition has a fasting plasma glucose level ⁇ 100 mg/dl and a two-hour post-glucose load plasma level ⁇ 140 mg/dl during an oral glucose tolerance test with 75 g glucose (in other terms, exhibits no prediabetes according to ADA definition); and, optionally displays a HbA1c ⁇ 5.7% and is representative (with regard to, e.g., same distribution of age, race, sex, health status) for the investigated population, and/or ii) database entries of corresponding groups of subjects as defined in i).
  • the method includes a further step to determine that the subject does not have T2D, wherein if the subject does not have T2D, the amount(s) of the protein(s) and/or mRNA(s) as specified in the claim are indicative of a predisposition to develop a cardiometabolic disease, namely in this embodiment T2D.
  • the method includes a further step to determine that the subject does not have prediabetes or T2D, wherein if the subject does not have prediabetes or T2D, the amount(s) of the protein(s) and/or mRNA(s) as specified in the claim are indicative of a predisposition to develop a cardiometabolic disease, namely in this embodiment prediabetes or T2D.
  • the method preferably, includes a further step to determine that the subject does not have a cardiometabolic disease, wherein if the subject does not have a cardiometabolic disease, the amount(s) of the protein(s) and/or mRNA(s) as specified in the claim are indicative of a predisposition to develop a cardiometabolic disease.
  • an “increase" in the amount of MASP-1 , GPLD1 , or THBS1 or the amount of an mRNA encoding MASP-1 , GPLD1 , or THBS1 in accordance with the invention is meant to refer to an increase of at least (for each value) 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% as compared to the amount of the respective controls.
  • an increase in the amount of MASP-1 refers to an increase of at least (for each value) 15%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40%, lower increased values being characteristic especially for prediabetes and higher increased values for type 2 diabetes, as compared to the MASP-1 amount of the control.
  • an increase in the amount of GPLD1 refers to an increase of at least (for each value) 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or at least 45%, lower values being characteristic especially of prediabetes and higher values of type 2 diabetes, as compared to the GPLD1 amount of the control.
  • an increase in the amount of THBS1 refers to an increase of at least (for each value) 15%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60%, lower increased values being characteristic especially of type 2 diabetes and higher increased values of prediabetes, as compared to the THBS1 amount of the control.
  • a “decrease” in the amount of APOA-IV or the amount of an mRNA encoding APOA-IV in accordance with the invention is meant to refer to a decrease of at least (for each value) 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared to the APOA-IV amount of the control.
  • Preferred values of decrease are at least 25% or at least 30%, especially for prediabetes.
  • the increase or decrease is a statistically significant increase or decrease, respectively, in comparison to the control which can be calculated by methods well-known in the art.
  • MASP-1 , GPLD1 , THBS1 and APOA-IV have been identified to be associated with the risk to develop a cardiometaboiic disease.
  • said association of MASP-1 , GPLD1 , THBS1 can be assessed in the form of an increase of the expression levels, both on the transcriptional level (mRNA) and the translational level (protein), of MASP-1 , GPLD1 , THBS1 in comparison to levels of these protein(s) or mRNA(s) of control subjects.
  • Said association of APOA-IV can be assessed in the form of a decrease in the expression level, both on the transcriptional level (mRNA) and the translational level (protein), of APOA-IV in comparison to expression levels of a control.
  • assessment of the expression levels of said markers in accordance with the invention is a valuable tool in the identification and subsequent supervision and care of subjects at risk of developing a cardiometaboiic disease.
  • the invention relates to a method of identifying a compound capable of preventing or treating a cardiometaboiic disease and diseases associated therewith or serving as a lead compound for developing a compound capable of preventing a cardiometaboiic disease and diseases associated therewith, the method comprising the steps of: (a) assessing the amount of one or more protein(s) and/or mRNAs encodings said protein(s), wherein said one or more protein(s) is/are selected from mannan-binding lectin serine peptidase 1 (MASP-1 ), glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1 ), thrombospondin (THBS1 ) and apolipoprotein A-IV (APOA-IV), in a cell contacted with a test compound or in a sample obtained from a subject and contacted with said test compound; and (b) assessing the amount of said one or more protein(s) or mRNA(MASP-1 ),
  • step (a) not contacted with said test compound; ii. contacted with a compound known to not affect the amount of said one or more protein(s) or mRNA(s) of step (a), wherein a decrease in the amount of MASP-1 , GPLD1 , or THBS1 or of an mRNA encoding MASP-1 , GPLD1 , or THBS1 or an increase in the amount of APOA-IV or of an mRNA encoding APOA-IV assessed in step (a) as compared to the amount(s) assessed in step (b) i. or (b) ii.
  • test compound being capable of preventing a cardiometaboiic disease and diseases associated therewith or serving as a lead compound for developing a compound capable of preventing a cardiometaboiic disease and diseases associated therewith; and/or iii. contacted with a known agent for preventing or treating a cardiometaboiic disease, wherein an essentially equal amount or a decrease of the amount of MASP-1 , GPLD1 , or THBS1 or of an mRNA encoding MASP-1 , GPLD1 , or THBS1 and/or an essentially equal amount or an increase in the amount of APOA-IV or of an mRNA encoding APOA-IV assessed in step (a) as compared to the amount(s) assessed in step (b) iii. is indicative of said compound being capable of preventing a cardiometaboiic disease and diseases associated therewith or serving as a lead compound for developing a compound capable of preventing a cardiometabolic disease and diseases associated therewith.
  • the amount of the one or more protein(s) or mRNA(s) is to be assessed in a cell or in a sample obtained from a subject contacted with the test compound.
  • Said cell may be part of a cell culture colony derived from an established cell line.
  • the cells could be hepatocytes, cardiomyocytes, adipocytes, myocytes, epithelial cells (of kidney, lung or cardiovascular origin) and/or fibroblasts, and cells derived from these (e.g. immortalized cell lines or induced pluripotent stem cell lines).
  • the cell may be part of a primary cell culture established from a sample of a subject contacted with the test compound. Suitable samples obtained or obtainable from subjects for the assessment of protein or mRNA amounts are described herein elsewhere and may also be processed without prior steps, such as establishing a primary cell culture, in order to assess protein or mRNA amounts.
  • the subject from which the sample or cell in steps (a) and (b) is obtained is the same and may be, e.g., a subject belonging to the control group defined herein above in relation to method for identifying a predisposition for developing a cardiometabolic disease in a subject, or for producing a diagnostically informative value.
  • the subject may be person identified or otherwise specified (e.g. due to being considered a disease model) to be at risk for developing a cardiometabolic disease.
  • the subject may in particular include animals such as, e.g., rodents (preferably mice, rats, rabbits), pigs, sheep and monkeys.
  • rodents preferably mice, rats, rabbits
  • pigs preferably sheep and monkeys.
  • the subject is a human subject.
  • compound as used herein relates to a substance that may be solid, semisolid, semifluid, fluid or gaseous. Said compound may, however, also be comprised in a mixture, extract or composition.
  • the compound that is identified according to the method of the invention to be capable of preventing a cardiometabolic disease and diseases associated therewith or capable of serving as a lead compound for developing a compound capable of preventing a cardiometabolic disease and diseases associated therewith decreases the amount of said one or more protein(s) selected from MASP-1 , GPLD1 , and THBS1 and said one or more mRNA(s) encoding said protein(s) and/or increase the amount of said APOA-IV or mRNA encoding APOA-IV, which may, for example, be based on its inhibitory, promoting, agonistic or antagonistic activity in a direct or indirect interaction with the given protein itself, with gene sequences involved in the generation of a given protein or mRNA or with any intermediate or final gene product(s).
  • Said compound(s) may be chemically synthesized or produced via microbial fermentation but can also be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
  • said compound to be identified by the method of the invention may be known in the art but hitherto not known to be useful as a compound capable of preventing a cardiometabolic disease.
  • said compound may display its decreasing or increasing activity, as the case may be, only with regard to one of the proteins or mRNAs analysed should more than one protein or its encoding mRNA be part of the screening method.
  • using more than one protein or mRNA encoding said more than one protein can be advantageous to identify compounds that display decreasing or increasing activity with regard to more than one protein or mRNA encoding said more than one protein simultaneously.
  • mixtures of several compounds or compositions comprising several compounds which can exhibit the desired increasing or decreasing activity with regard to (a) different protein(s) or mRNA(s).
  • Diseases associated with a cardiometabolic disease are diseases whose onset and/or persistence are causally related to the development and/or presence of a cardiometabolic disease or have been shown to develop and persist simultaneously with a cardiometabolic disease (such as e.g., in the case of type 2 diabetes, cardiovascular diseases (e.g., stroke, heart attack), diabetic retinopathy and neuropathy, or kidney failure). Also envisaged is that diseases associated with a cardiometabolic disease are diseases that the subject is afflicted with prior to developing a cardiometabolic disease and that are modulated such as, e.g., exacerbated or shifted to a chronic status from an acute status upon development and/or presence of a cardiometabolic disease.
  • the interaction may take place extracellularly and/or intracellularly resulting in the decrease of the amount of one or more protein(s) selected from MASP-1 , GPLD1 , and THBS1 and said one or more mRNA(s) encoding said protein(s) and/or increase the amount of said APOA-IV or mRNA encoding APOA-IV - provided the compound is capable thereof.
  • a compound may bind to the cell surface and trigger a signal cascade resulting in an increase or decrease of said protein or mRNA amounts.
  • the compound is preferably exposed to the cell under conditions allowing both extracellular interaction and the uptake into the cell, in particular the cytoplasma or nucleus, to exert its potential activity on regulatory events in the expression of a given protein.
  • Conditions generally suitable for uptake of compounds such as, e.g., proteins or nucleic acid molecules, into cells and methods to enhance said uptake as regards rate and amount wherein said enhancement may include artificially modifying, e.g, proteins, are known in the art (see, for example, Patsch and Edenhofer, (2007), Handb. Exp.
  • cell lines naturally exhibiting the capacity of increased uptake capabilities relative to other cells are also known in the art. Such cells are, for example, cells like mucosal cells or intestinal cells.
  • a number of mechanisms exist for the passage of various compounds across the plasma membrane including passive diffusion, facilitated diffusion, and active transport systems. Passive diffusion of proteins through the bilayer lipid structure of the plasma membrane is a function of the size, lipid solubility, and charge of the protein molecule.
  • a further uptake mechanism is endocytosis. Endocytosis is a process whereby cells absorb material from the outside by engulfing it with their cell membrane.
  • test compound should be contacted with the subject in a way that allows for the interaction of the test compound with cells that are part of the sample to be subsequently obtained from said subject and assessed in accordance with the method of the invention.
  • intravenous administration of the test compound - if necessary as part of a therapeutically acceptable composition - will be suitable to allow for an interaction of cells of the sample with the test compound.
  • said test compound may be, e.g., added to the culture medium or injected into a cell or administered to an individual prior to the assessment of the protein or mRNA amounts in step (a).
  • the compound to be identified can be contained in libraries of small molecules, such as organic or inorganic small molecules which may be commercially available.
  • libraries comprising antibodies or functional fragments or derivatives thereof (i.e. fragments or derivatives maintaining the binding specificity of the original antibody) may be used as a starting point in the identifying process.
  • Suitable libraries are commercially available, for example from ChemBridge Corp., San Diego, USA.
  • libraries of aptamers such as peptide aptamers might be employed. The skilled person is, of course free, to use any other starting point of desired compounds for use in the method of the invention.
  • composition containing (a) compound(s) is identified to be capable of preventing a cardiometabolic disease and diseases associated therewith or serving as a lead compound for developing a compound capable of preventing a cardiometabolic disease or diseases associated therewith in the method of the invention, then it is either possible to isolate the active compound(s) from the original composition identified as containing the compound(s) in question or one can further subdivide the original composition, for example, if it consists of a plurality of different test compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original composition.
  • compositions or resulting compound displays the desired properties, for example, by the methods described herein or in the literature ("Cells: A laboratory manual", v. 1-3, edited by Spector et al., Cold Spring Harbour Laboratory Press (1997); ISBN 10: 0879695218).
  • the steps described above can be performed several times, preferably until the composition identified according to the method of the invention only comprises a limited number of or only one substance(s).
  • said composition comprises substances of similar chemical and/or physical properties.
  • the method of the present invention can be easily performed and the experimental setup without further ado designed by the person skilled in the art, for example, in accordance with other cell based screening assays described in the prior art.
  • Compounds which can be tested in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like.
  • said compounds may act as antagonists or agonists, respectively.
  • Said compounds can also be functional derivatives or analogues of known drugs.
  • Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.
  • peptide mimetics and/or computer-aided design of appropriate drug derivatives and analogues can be used.
  • Appropriate computer programs can be used for the identification of interactive sites of a compound putatively capable of preventing a cardiometabolic disease and diseases associated therewith by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 1 14-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991.
  • results obtained from the above-described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known inhibitors, analogs, antagonists or agonists.
  • Appropriate peptidomimetics can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described or referred to herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
  • the three-dimensional and/or crystallographic structure of said compounds can be used for the design of peptidomimetic drugs (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545- 1558). It is well known how to obtain compounds to be tested in the method of the invention, e.g. by chemical or biochemical standard techniques. Thus, also comprised by the method of the invention are means of making or producing said compounds. In summary, the present invention provides a method for identifying compounds which can be used for the prevention of a cardiometabolic disease and diseases associated therewith.
  • the method of the invention may be useful in identifying lead compounds.
  • the term "lead compound" in accordance with the present invention refers to a compound discovered by the method of the invention which will be e.g. further optimized, in particular to be pharmaceutically more acceptable.
  • the identified lead compounds may be optimized to arrive at a compound which may be, for example, used in a pharmaceutical composition for preventing a cardiometabolic disease.
  • Methods for the optimization of the pharmacological properties of compounds identified in screening methods such as the screening method according to the invention, the lead compounds, are known in the art and comprise a method of modifying a compound identified as a lead compound to achieve: (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carboxylic acids
  • a Pope-prodrug is a compound that is generally not biologically and/or pharmacologically active. After administration, the prodrug is activated, typically in vivo by enzymatic or hydrolytic cleavage and converted to a biologically and/or pharmacologically compound which has the intended medical effect.
  • Prodrugs are typically formed by chemical modification of biologically and/or pharmacologically compounds. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.
  • a corresponding formulation as a prodrug can be used which is converted into its active in the patient.
  • Precautionary measures that may be taken for the application of prodrugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329.
  • the screening method is an in vitro or ex vivo method.
  • said method is effected in high-throughput format.
  • High- throughput assays independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices.
  • mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well.
  • said mixture of test compounds may be de-convoluted to identify the one or more test compounds in said mixture giving rise to said activity.
  • human cells are envisaged for use in any of the methods of the invention, it is preferred that such human cells are not obtained from a human embryo, in particular not via methods entailing destruction of a human embryo.
  • human embryonic stem cells are at the skilled person's disposal such as taken from existent embryonic stem cell lines commercially available.
  • the present invention may be worked with human embryonic stem cells without any need to use or destroy a human embryo.
  • pluripotent cells that resemble embryonic stem cells such induced pluripotent stem (iPS) cells may be used, the generation of which is state of the art (Hargus G et al., Proc Natl Acad Sci U S A 107:15921-15926; Jaenisch R. and Young R., 2008, Cell 132:567-582; Saha K, and Jaenisch R., 2009, Cell Stem Cell 5:584-595).
  • This method of the invention is based upon the plausible presumption that the decrease of the amount of said one or more protein(s) selected from MASP-1 , GPLD1 , and THBS1 or said one or more mRNA(s) encoding said protein(s) and/or increase the amount of said APOA-IV or mRNA encoding APOA-IV is suitable to prevent the development of a cardiometabolic disease and diseases associated therewith in a subject.
  • said protein(s) or mRNA(s) encoding said one or more protein(s) is/are associated with the risk of developing a cardiometabolic disease and their amounts are either increased or decreased, respectively, in subjects at risk for developing a cardiometabolic disease.
  • the increase and/or decrease in protein or mRNA amount(s) is determined vis-a-vis the corresponding protein or mRNA amount(s) of a control sample or control cell in step (b).
  • control samples of step (b) i), ii) or iii) are not to be contacted with the test compound referred to in step (a).
  • said control sample or cell may be a sample or cell that has not been contacted with the compound to be tested (step (b)i.).
  • the protein or mRNA amount(s) of the sample or cell may be compared to the corresponding protein or mRNA amount(s) of a sample or cell that has been contacted with a compound known to not affect the amount(s) of said protein(s) or mRNA(s) (step (b)ii.). It is also envisaged that in addition to the comparison of the protein or mRNA amount(s) of the cells or the sample of step (a) against the one or both latter negative controls (step (b)i.
  • step (b)ii.) a comparison against a sample or cell contacted with a compound known to affect the amount of said one or more protein(s) or mRNA(s), in the case of MASP-1 , GPLD1 , or THBS1 to decrease the amount and in the case of APOA-IV to increase the amount, is effected.
  • a corresponding method provides a qualitative assessment of the compound to be tested in the sense that its activity is compared to a positive standard/control.
  • the different controls i.e. step (b) i., ii. and Hi., can be combined so that all three controls are used or, alternatively, only two only two controls are combined, preferably, a negative control and a positive control, i.e.
  • steps (b) i. or (b)ii., and (b)iii. are performed. It is possible to exclusively compare protein or mRNA amount(s) of the cell or sample of step (a) against the corresponding protein or mRNA amount(s) of a cell or sample that has been contacted with a compound known to affect the amount of said one or more protein(s) or mRNA(s), in the case of MASP-1 , GPLD1 , or THBS1 to decrease the amount and in the case of APOA- IV to increase the amount, if it is desired to screen for compounds that have a similar or superior effect on said amount(s) as the latter control.
  • a corresponding experimental setup would hence allow identifying compounds that are similar or superior to existing compounds that are capable of decreasing the amount of said one or more protein(s) selected from MASP-1 , GPLD1 , and THBS1 or said one or more mRNA(s) encoding said protein(s) and/or increasing the amount of said APOA-IV or mRNA encoding APOA-IV.
  • corresponding compounds may be capable of effecting an essentially equal or higher decrease of the amounts of the amount of said one or more protein(s) selected from MASP- 1 , GPLD1 , and THBS1 said one or more mRNA(s) encoding said protein(s) and/or an essentially equal or higher increase of the amount of said APOA-IV or mRNA encoding APOA-IV as compared to said compound known to decrease the amount of said one or more protein(s) selected from MASP-1 , GPLD1 , and THBS1 or said one or more mRNA(s) encoding said protein(s) and/or increase the amount of said APOA-IV or mRNA encoding APOA-IV.
  • the term "essentially equal” refers to (a) protein or mRNA amount(s) that differ(s) by at most (for each value) +/- 10%, +/- 9%, +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, +/- 2%, +/- 1 % from or is equal to the corresponding protein or mRNA amount(s) of the cell or sample that has been contacted with the compound known to decrease the amount of said one or more protein(s) selected from MASP-1 , GPLD1 , and THBS1 or said one or more mRNA(s) encoding said protein(s) or known to increase the amount of said APOA-IV or mRNA encoding APOA-IV.
  • the protein or mRNA amount(s) could be decreased or increased to a greater extent than the compound known to decrease or increase, respectively.
  • the increase or decrease can exceed the amounts of said compound known to decrease or increase by at least 11% such as, e.g., at least (for each value) 12%, 13%, 14%, 15%, or, preferably, at least 16% or at least 17%, more preferred at least 20% or at least 30% and most preferred at least 100%, such as at least 150% or at least 200%.
  • suitable compounds to be identified by the method of the invention may in comparison only achieve a fraction of the decrease and/or increase of the amount of said one or more marker(s) of the above defined positive control.
  • the identification of compounds that are capable of adjusting the amount of the risk marker(s) as described herein towards the amount of the corresponding marker(s) in the control (which is not at risk to develop a cardiometabolic disease), preferably selectively and in a dose-dependent fashion, provides the means for a drug-based therapeutic intervention.
  • the method further comprises the step of preparing a hard and/or soft copy comprising the values of amounts of protein(s) and/or mRNA(s) determined.
  • a hard and/or soft copy comprising the values of amounts of (a) protein(s) or mRNA(s) determined is prepared.
  • the hard or soft copy may comprise the values of the assessed amounts of the sample from the subject as well as the control.
  • the information produced by the method is in this preferred further step transformed to produce said soft and/or hard copy.
  • Non-limiting examples of hard copies include print-outs, hand-written information as well as photographs or the data as originally obtained, for example from a mass spectrometer.
  • Non-limiting examples of soft copies include any form of computer files such as the originally obtained data output from the machine performing the measurements (e.g. a mass spectrometer) or from the respective analysis program or e.g. word or other text software documents containing the values, as well as e.g. screen shots.
  • the values comprised in said hard or soft copy can e.g. be calculated values, for example in the form of numerical values derived from the measurements as well as the original data as obtained.
  • the preferred further step of preparing a hard or soft copy comprising said values of amounts can be applied to either the method of identifying a predisposition for developing a cardiometabolic disease of the present invention, or to the method for producing diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease of the invention.
  • the method further comprises synthesizing said compound being capable of preventing a cardiometabolic disease and diseases associated therewith or serving as a lead compound for developing a compound capable of preventing a cardiometabolic disease and diseases associated therewith.
  • the cardiometabolic disease is selected from type 2 diabetes mellitus, hypertension, myocardial infarction and stroke, acute coronary syndrome and metabolic syndrome.
  • the cardiometabolic disease is selected from type 2 diabetes mellitus.
  • Type 2 diabetes mellitus as used herein relates to a condition characterized by the development of the symptoms of increased blood glucose levels believed to be the result of beta-cell dysfunction and insulin resistance.
  • T2D can be preceded by a state termed “prediabetes” (also referred to in the art as “intermediate hyperglycemia”, “impaired glucose metabolism” or “impaired glucose regulation”) which is characterized by elevated blood glucose levels alone and/or the incapability to efficiently clear glucose from the blood stream as mentioned herein above; see, e.g., Menke et al., JAMA 314, 1021-1029 (2015).
  • prediabetes includes the physiological states i-IFG (isolated-impaired fasting glucose) and IGT (impaired glucose tolerance) as referred to in the below table based on WHO criteria.
  • ITT impaired glucose tolerance
  • a prediabetic subject may alternatively display only an elevated fasting blood glucose level and is then classified as having isolated- impaired fasting glucose (i-IFG): with a fasting glucose value of 110 ⁇ and ⁇ 126 mg/dl, but two-hour post glucose plasma load of ⁇ 140 mg/dl.
  • i-IFG isolated- impaired fasting glucose
  • Table 1 provides an overview of the definition of i-IGF and IGT, as referred to in accordance with the invention, which are approved by the World Health Organization (World, H.O. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1 : Diagnosis and classification of diabetes mellitus. Report of a WHO consultation (1999)).
  • an association of the amount of each of the proteins MASP-1 , GPLD1 , THBS1 and APOA-IV with prediabetes could be demonstrated.
  • the association could be demonstrated to be independent from the common risk factors of age, sex, BMI, smoking status, alcohol intake, physical inactivity, actual hypertension, triacylglycerol, total/HDL cholesterol ratio and C-reactive protein.
  • a screening and quantification of various pre-selected proteins was performed in a large cohort. The results of this study showed, inter alia, a strong and novel association between MASP-1 , GPLD1 , THBS1 and APOA-IV and prediabetes (see Examples and Figures).
  • these proteins and also the mRNA molecules encoding said proteins are each diagnostic markers for prediabetes. Since it is well-known that the status of prediabetes is associated with a risk to develop type 2 diabetes mellitus, each of these markers is also a prognostic marker for the development of T2D. It was not known prior to the invention that these proteins or mRNAs encoding said proteins can be used alone or in combination for the diagnosis of prediabetes and therefore for the prognosis of T2D. In particular, the association with prediabetes is independent from other common risk factors for T2D. As such, they represent valuable prognostic markers in particular for the development of T2D, besides other cardiometabolic diseases.
  • the method is also a method of diagnosing prediabetes characterized by the assessment step described herein above, wherein an increase in the amount of MASP-1 , GPLD1 , or THBS1 or an mRNA encoding MASP-1 , GPLD1 , or THBS1 or a decrease in the amount of APOA-IV or an mRNA encoding APOA-IV as compared to the amount of said protein(s) or said mRNA(s) of a control is not only indicative of a predisposition to develop type 2 diabetes mellitus, but also indicative of prediabetes, i.e. the subject has prediabetes.
  • the present invention also provides a method of diagnosing prediabetes in a subject, said method comprising or consisting of the step of assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from mannan-binding lectin serine peptidase 1 (MASP-1 ), glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1 ), thrombospondin (THBS1 ) and apolipoprotein A-IV (APOA-IV), wherein an increase in the amount of MASP-1 , GPLD1 , or THBS1 or of an mRNA encoding MASP-1 , GPLD1 , or THBS1 or a decrease in the amount of APOA-IV or of an mRNA encoding APOA-IV as compared to the amount of said protein(s)
  • the present invention also provides a method of diagnosing prediabetes in a subject, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amount of one or more protein(s) and/or mRNA(s) encoding said protein(s), wherein said one or more protein(s) is/are selected from a group of proteins as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of APOC2, APOE, CRP, LBP, MASP-1 , ADIPOQ, APOA-IV, SHBG and optionally furthermore APOC3; (b) said parameters are age, sex and BMI, and said group consists of APOC3, APOE, MASP-1 , ADIPOQ, PZP and SHBG or said group consists of APOA-IV; or (c) said parameters are age, sex, BMI, smoking status, alcohol
  • the present invention also provides a method of diagnosing prediabetes in a subject, said method comprising or consisting of the step of (i) assessing in a sample obtained from said subject the amounts of all members of a group of proteins and/or mRNAs encoding said proteins, wherein said group of proteins is as defined in the following, and (ii) determining parameters of said subject, wherein (a) said parameters are age and sex, and said group consists of APOC3, C7, CD14, LBP, MASP-1 , ADIPOQ, APOA-IV, ITIH4 and SHBG or consists of C7, CRP, GPLD1 , LBP, MASP-1 , APOA-IV, ORM1 and SHBG; (b) said parameters are age, sex and BMI, and said group consists of APOE, CD14, CD5L, LBP, ADIPOQ, PZP and SHBG or consists of C7, LBP, MASP-1 , APOA-IV, OR
  • the first groups in items (a), (b) and (c) relate to the definition of prediabetes according to ADA, the second groups according to WHO.
  • control for these embodiments is preferably selected from i) a group of subjects (termed herein also "control group") negative for T2D, exhibiting no impaired glucose tolerance (IGT) and, exhibiting no isolated impaired fasting glucose (i-IFG), i.e.
  • control group according to WHO definition has a fasting plasma glucose level ⁇ 110 mg/dl and a two-hour post-glucose load plasma level ⁇ 140 mg/dl during an oral glucose tolerance test with 75 g glucose (in other terms, exhibits no prediabetes according to WHO definition);
  • control group according to ADA definition has a fasting plasma glucose level ⁇ 100 mg/dl and a two-hour post-glucose load plasma level ⁇ 140 mg/dl during an oral glucose tolerance test with 75 g glucose (in other terms, exhibits no prediabetes according to ADA definition), and, optionally displays a HbA1c ⁇ 5.7% and is representative (with regard to, e.g., same distribution of age, race, sex, health status) for the investigated population, and/or ii) database entries of corresponding groups of subjects as defined in i).
  • the method includes a further step to determine that the subject does not have T2D, wherein if the subject does not have T2D, the amount(s) of the protein(s) and/or mRNA(s) as specified in the embodiments relating to diagnosis of prediabetes are indicative to diagnose a cardiometabol ' ic disease, namely in this embodiment prediabetes.
  • the cardiometabolic disease is type 2 diabetes mellitus.
  • the amount of two or more proteins and/or mRNAs is assessed; or the amount of three or more proteins and/or mRNAs is assessed.
  • the prognostic value can be increased by assessing more than one of these markers, i.e. a combination.
  • a combination is specific combinations of markers that together provide for a particularly improved prediction of the risk to develop a cardiometabolic disease, in particular the risk to develop T2D.
  • the amount of MASP-1 in combination with one of GPLD1 , THBS1 and APOA-IV or mRNAs encoding said proteins is assessed; the amount of MASP-1 and GPLD1 or mRNAs encoding said proteins, the amount of MASP-1 and THBS1 or mRNAs encoding said proteins; the amount of MASP-1 and APOA-IV or mRNAs encoding said proteins, the amount of GPLD1 and THBS1 or mRNAs encoding said proteins, the amount of GPLD1 and APOA-IV or mRNAs encoding said proteins, or the amount of THBS1 and APOA-IV or mRNAs encoding said proteins is assessed; the amount of MASP-1, GPLD1 and TH
  • the amount of one or more protein(s) or mRNA(s) encoding said protein(s) is assessed, wherein said one or more protein(s) is/are selected from CD5 antigen-like protein (CD5L), gelsolin (GSN), inter-alpha-trypsin inhibitor heavy chain H4 protein (ITIH4), apolipoprotein E (APOE), tetranectin (CLEC3B), C-reactive protein (CRP), lipopolysaccharide binding protein (LBP), mannose-binding lectin 2 (MBL2), and/or pregnancy zone protein (PZP), and wherein a modulation of the amount of CD5L, GSN,
  • the protein (SEQ ID NO: 9) and cDNA sequence (SEQ ID NO: 10) of CD5L are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000357156 and ENST00000368174, respectively.
  • the protein (SEQ ID NO: 11 ) and cDNA sequence (SEQ ID NO: 12) of GSN are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000362924 and ENST00000373818, respectively.
  • the protein binds to the "plus" ends of actin monomers and filaments to prevent monomer exchange.
  • the protein (SEQ ID NO: 13) and cDNA sequence (SEQ ID NO: 14) of ITIH4 are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000417824 and ENST00000485816, respectively.
  • the protein is secreted into the blood, where it is cleaved by plasma kallikrein into two smaller forms.
  • the protein (SEQ ID NO: 15) and cDNA sequence (SEQ ID NO: 16) of APOE are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000252486 and ENST00000252486, respectively.
  • the protein is a major apoprotein of the chylomicron.
  • the protein (SEQ ID NO: 17) and cDNA sequence (SEQ ID NO: 18) of CLEC3B are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1)) under accession number ENSP00000296130 and ENST00000296130, respectively.
  • the protein (SEQ ID NO: 19) and cDNA sequence (SEQ ID NO: 20) of CRP are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1)) under accession number ENSP00000255030 and ENST00000255030, respectively.
  • the protein is involved in several host defense related functions based on its ability to recognize foreign pathogens and damaged cells of the host and to initiate their elimination by interacting with humoral and cellular effector systems in the blood.
  • the protein (SEQ ID NO: 21) and cDNA sequence (SEQ ID NO: 22) of LBP are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1)) under accession number ENSP00000217407 and ENST00000217407, respectively.
  • the protein is involved in the acute-phase immunologic response to gram-negative bacterial infections.
  • the protein (SEQ ID NO: 23) and cDNA sequence (SEQ ID NO: 24) of MBL2 are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1)) under accession number ENSP00000363079 and ENST00000373968, respectively.
  • the protein belongs to the collectin family and is an element in the innate immune system.
  • the protein recognizes mannose and N-acetylglucosamine on many microorganisms, and is capable of activating the classical complement pathway.
  • the protein (SEQ ID NO: 25) and cDNA sequence (SEQ ID NO: 26) of PZP are known in the art and can be found in the EnsembI Human database (release 72 (GRCm38.p1 )) under accession number ENSP00000261336 and ENST00000261336, respectively.
  • the amount of proteins GSN, ITIH4, APOE, CLEC3B, CRP, LBP, MBL2 or mRNAs encoding said proteins are increased, whereas the amount of proteins CD5L, PZP or mRNAs encoding said proteins are decreased in comparison to the corresponding amounts of a control to be considered indicative of a predisposition to develop a cardiometabolic disease.
  • a predisposition for developing a cardiometabolic disease in a subject or for producing a diagnosticaily informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease
  • in addition to the amount of MASP-1 or an mRNA encoding MASP-1 the amount of APOE, CRP and ITIH4 or mRNAs encoding APOE, CRP and ITIH4 is assessed; in addition to the amount of MASP-1 or an mRNA encoding MASP-1 the amount of APOE, CRP, LBP and CD5L or mRNAs encoding APOE, CRP, LBP and CD5L is assessed; or in addition to the amount of MASP-1 or an mRNA encoding MASP-1 the amount of LBP, CLEC3B, CRP, GSN, MBL2 and PZP or mRNAs encoding L
  • the combination of the markers MASP-1 , GPLD1 , THBS1 and APOA-IV and all additional markers provides an enhanced predictive value, in particular when adjusted for the risk factors age, sex, BMI, smoking status, alcohol intake, physical inactivity, actual hypertension, triacylglycerol and total cholesterol/ HDL cholesterol ratio.
  • Table 2 depicts different multivariable models calculated presenting effect estimates of the respective combination of candidates including statistical significance.
  • the area under the curve value (AUC) indicates the predictive power after inclusion of protein markers (AUC Extended) compared to the predictive power of only the other adjusted risk factors (AUC Base). The higher the AUC value is, the higher is the predictive power. The higher the difference between AUC Extended and AUC Base is, the higher is the gain in prediction by inclusion of the proteins.
  • Table 2a relates to the data set referred to as "lot 1" in the examples, and Table 2b to the "lot 1 and lot 2 combined" data set.
  • Table 2a Lot 1 multimarker proteomics results for the response 'prevalent prediabetes WHO definition' according to different protein and adjustment models model 3 with simultaneous inclusion of 13 proteins : AUC AUC adjusted for age, sex, BMI, smoking status, alcohol intake, physical Base Extended
  • model 2b with simultaneous inclusion of five proteins: AUC AUC adjusted for age, sex, BMI, smoking status, alcohol intake, physical Base Extended
  • model 2 with simultaneous inclusion of five proteins Base Extended adjusted for age, sex, BMI (without (with proteins) proteins)
  • model 1 Base Extended adjusted for age and sex (without (with proteins) proteins)
  • model 1 Base Extended adjusted for age and sex (without (with proteins) proteins)
  • model 1 Base Extended adjusted for age and sex (without (with proteins) proteins)
  • model 1 Base Extended adjusted for age and sex (without (with proteins) proteins)
  • model 1 Base Extended adjusted for age and sex (without (with proteins) proteins)
  • the predictive value of the method of the invention can be even further enhanced, if the amount(s) of the following one or more additional protein(s) or mRNA(s) is/are assessed, wherein said one or more protein(s) is/are selected from adiponectin (ADIPOQ), apolipoprotein C-ll (APOC2), apolipoprotein C-lll (APOC3), complement component 7 (C7), complement component 8, alpha polypeptide (C8A), complement component 8, beta polypeptide (C8B), CD14 molecule (CD14), orosomucoid 1 (ORM1 ), retinol binding protein 4, plasma (RBP4) and/or sex hormone-binding globulin (SHBG), and wherein an increase or decrease in the amount of ADIPOQ, APOC2, APOC3, C7, C8A, C8B, CD14, ORM1 , RBP4 or SHBG or an mRNA encoding said one or more protein(s) as compared to the
  • the sample is selected from blood, serum or plasma.
  • One preferred sample to be used in the methods of the invention is blood (cf. also supra) due to the ease of accessibility and clinical routine of blood collection as well as the standardized protocols to extract and, optionally, purify proteins or mRNAs from blood constituents.
  • Preferred in accordance with the method of the invention are peripheral blood mononuclear cells as a blood constituent that is to be further processed for extraction of proteins or mRNAs.
  • Serum or plasma as sample is also preferred. Suitable methods for extraction and optional purification of proteins or mRNAs from blood, serum or plasma are well-known to the person skilled in the art and exemplary methods are described herein.
  • sample in accordance with the methods of the invention are saliva and buccal smear samples that are equally conveniently accessible for collection, however, without having to injure a subject in order to get to the sample as compared to blood, serum or plasma collection.
  • buccal mucosa epithelial cells that can be isolated from buccal swabs for further processing to extract and, optionally, purify proteins or mRNAs in order to assess amounts of the markers associated with the risk to develop a cardiometabolic disease.
  • said assessment of the amount of said one or more protein(s) is effected by targeted selected reaction monitoring (SRM) tandem MS.
  • SRM-MS is a preferred assessment method for determining/assessing the amount of said one or more protein(s).
  • the mass spectrometry based method of SRM-MS has been described herein above.
  • antibody based methods for protein measurement are known to involve time-consuming, costly, and failure prone development.
  • SRM-MS overcomes these limitations. Unlike ELISA, SRM assays can be generated for any detectable protein in a tailored fashion, further allowing multiplexing to a high degree (see Picotti et al., Nat Methods. 2013 Jan;10(1 ):24-7).
  • said test compound is selected from a small molecule, an antisense nucleic acid molecule, a siRNA, a shRNA, a miRNA, a ribozyme, a peptide aptamer, a nucleic acid based aptamer, an antibody or a combination thereof.
  • small molecule as used herein may describe, for example, a small organic molecule.
  • Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds.
  • the original definition of the term organic related to the source of chemical compounds with organic compounds being those carbon-containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources.
  • Organic compounds can be natural or synthetic.
  • the compound may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates).
  • the small molecule has a molecular weight of less than about 2000 amu, or less than about 1000 amu such as 500 amu, and even less than about 250 amu.
  • the size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. Small molecules may be designed, for example, in silico based on the crystal structure of potential drug targets, where sites presumably responsible for the biological activity and involved in the modulation of protein or mRNA amount(s) identified herein, can be identified and verified in in vivo assays such as in vivo HTS (high-throughput screening) assays.
  • antisense nucleic acid molecule refers to a nucleic acid which is complementary to a target nucleic acid.
  • An antisense molecule is capable of interacting with, more specifically hybridizing with the target nucleic acid. By formation of the hybrid, transcription of the targeted gene(s) and/or translation of the targeted mRNA is reduced or blocked.
  • the antisense nucleic acid molecule is an antisense RNA molecule. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991 ) 51 :2897-2901 ).
  • RNA inactivation by antisense molecules or by ribozymes appears to be implementable. Both classes of compounds can be synthesized chemically or produced in conjunction with a promoter by biological expression in vitro or even in vivo.
  • siRNA Small interfering RNAs
  • siRNA small interfering RNAs
  • RNAi RNA interference pathway
  • siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.
  • Said siRNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • at least one RNA strand has a 5'- and/or 3'-overhang.
  • one end of the double-strand has a 3'-overhang from 1-5 nucleotides, more preferably from 1-3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3'-overhang.
  • any RNA molecule suitable to act as siRNA is envisioned in the present invention.
  • siRNAs have a well defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (- OH) group.
  • dsRNA short double-strand of RNA
  • - OH 3' hydroxyl
  • siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • siRNA duplexes composed of 21 -nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3'- overhang.
  • the sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et a/., EMBO J 2001 , 20(23):6877-6888).
  • 2 -deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant.
  • shRNA short hairpin RNA
  • RISC RNA-induced silencing complex
  • Si/shRNAs to be used in the method of the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL , USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • siRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs.
  • the RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi.
  • RNAi RNA-associated RNAs
  • Said RNA species are single-stranded RNA molecules which as endogenous RNA molecules regulate gene expression.
  • Upon binding to a complementary mRNA transcript triggers the degradation of said mRNA transcript through a process similar to RNA interference.
  • miRNAs may be employed to directly or indirectly regulate the amounts of proteins or mRNAs described herein to be associated with the risk to develop a cardiometabolic disease as defined herein.
  • ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a variety of reactions. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.
  • RNAs examples include the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes.
  • the organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.
  • the principle of catalytic self-cleavage has become well established in the last 10 years.
  • the hammerhead ribozymes are characterized best among the RNA molecules with ribozyme activity.
  • hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
  • the basic principle of constructing hammerhead ribozymes is as follows: An interesting region of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. Molecules of this type were synthesized for numerous target sequences. They showed catalytic activity in vitro and in some cases also in vivo. The best results are usually obtained with short ribozymes and target sequences.
  • Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Further, they can be combined with ribozymes to self-cleave in the presence of their target molecule.
  • aptamers can be classified as DNA or RNA aptamers or peptide aptamers. Whereas the former consist of (usually short) strands of oligonucleotides, the latter consist of a short variable peptide domain, attached at both ends to a protein scaffold.
  • Nucleic acid aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Peptide aptamers are proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range).
  • the variable loop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein which has good solubility properties.
  • the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge.
  • Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system.
  • Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight.
  • Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging.
  • Several modifications, such as 2'- fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, etc. are available to scientists with which the half-life of aptamers easily can be increased to the day or even week time scale.
  • a recent development is the combination of an aptamer recognizing a small compound with a hammerhead ribozyme.
  • the conformational change induced in the aptamer upon binding the target molecule is supposed to regulate the catalytic function of the ribozyme.
  • antibody as used herein can be, for example, relate to polyclonal or monoclonal antibodies.
  • antibody also comprises derivatives or fragments thereof with retained binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999.
  • the antibodies can be used in accordance with the method of the invention to modulate the amount of any metabolite associated with a risk to develop T2D described herein.
  • antibodies may be useful, for example, for immunoprecipitation, affinity purification and immunolocalization of the proteins associated with a risk to develop a cardiometabolic disease as well as for the monitoring of the presence and amount of such proteins, for example, in cultures of eukaryotic cells or organisms.
  • an antibody to be used in accordance with the invention also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanized (human antibody with the exception of non-human CDRs) antibodies, as well as antibody fragments, like, inter alia, Fab or Fab' fragments.
  • Antibody fragments or derivatives further comprise Fd, F(ab')2, Fv or scFv fragments; see, for example, Harlow and Lane (1988) and (1999), loc. cit.
  • the (antibody) derivatives can be produced by peptidomimetics.
  • Examples for such techniques include the hybridoma technique (Kohler and Milstein, Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).
  • Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of marker protein described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol.
  • antibody comprises antibody constructs which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, amongst others, viruses or plasmid vectors.
  • the antibody to be used in the method of the invention is capable to specifically bind/interact with a target epitope.
  • the term "specifically binding/interacting with” as used in accordance with the present invention means that the antibody does not or essentially does not cross-react with an epitope of similar structure.
  • Cross-reactivity of a panel of antibodies under investigation may be tested, for example, by assessing binding of said panel of antibodies under conventional conditions to the epitope of interest as well as to a number of more or less (structurally and/or functionally) closely related epitopes. Only those antibodies that bind to the epitope of interest in its relevant context (e.g.
  • protein and mRNA amounts can be significantly influenced by targeting, e.g., regulatory or enzymatically active proteins in the transcriptional or translational pathway of a given marker identified herein as risk factor for developing a cardiometabolic disease or said markers directly. Therefore, it will be possible to decrease or increase the amount of said given marker by using, e.g., the above described antisense nucleic acid molecule, the RNA molecules or antibodies targeting said markers directly or regulatory or enzymatically active proteins or the genes encoding them.
  • Said compounds may be designed by methods well-known in the art to interact target-specifically with either mRNA or proteins or with mRNA or protein molecules of said regulatory or enzymatically active proteins and thereby, indirectly, reduce the mRNA or protein amounts of said one or more markers identified herein as risk factors.
  • RNA molecules suitable for RNA interference can be designed leading to a reduction of mRNA molecules and hence also of the expression level of the marker proteins.
  • Proteins may also be directly targeted when using, e.g. antibodies, to decrease the amount of a protein selected from ASP-1 , GPLD1 and THBS1.
  • an increase or decrease in the amount of a given protein or mRNA may (only) be achievable when combining several (different) of the above described compounds.
  • a combination of said test compounds are used in the method of the invention and contacted simultaneously with a cell or a test sample obtained from a subject.
  • the invention further relates to a method of selecting a therapy to prevent a cardiometabolic disease for example T2D, comprising the steps of: (a) identifying a predisposition for developing a cardiometabolic disease in a subject or producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease according to the method of the invention, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease; and (b) selecting a therapy based on the results obtained in the preceding step.
  • the knowledge deduced from the present invention can now be used to exactly and reliably characterize the protein or mRNA profile of a subject as far as it is relevant in the identification of a predisposition to develop a cardiometabolic disease, preferably T2D.
  • a cardiometabolic disease preferably T2D.
  • the development of a cardiometabolic disease can be predicted and preventive measures can be applied accordingly.
  • a suitable individual therapy can be designed based on the knowledge of the individual protein profile of a subject with respect to the protein(s) or mRNA(s) indentified herein to be associated with a risk to develop a cardiometabolic disease and new and/or improved therapeutics can be identified, for example, by the method of the invention, and/or developed as has been discussed supra.
  • the method of identifying a predisposition for developing a cardiometabolic disease in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease can comprise a further step of applying a therapy to prevent a cardiometabolic disease, in case a predisposition to develop a cardiometabolic disease is identified.
  • the cardiometabolic disease to be identified and to be prevented is type 2 diabetes meliitus.
  • a suitable medical intervention such as, e.g., a drug treatment and/or changes in lifestyle (such as, e.g., dietary changes), having overall a more beneficial effect than medical approaches without having regard to the individual profile of the proteins or mRNAs associated with a risk to develop a cardiometabolic disease.
  • the effects of therapies e.g., pharmacologic effects of drugs, can be determined by methods well-known in the art and include, for example, in vitro methods or collecting data relating to disease symptoms in a patient or group of patients.
  • said method comprises i) prior to step (b) a further step (a') of monitoring an applied therapy; and/or ii) after step (b) a further step (b') of monitoring the therapy selected in step (b).
  • the method of selecting a therapy may comprise the additional step (a') of monitoring an applied therapy and based on its outcome select a therapy.
  • the method of selecting a therapy may also include applying the selected therapy.
  • none of the embodiments of this method of the invention include the actual application of a therapy, i.e. the method does preferably not include methods for the treatment of the human or animal body by surgery or therapy.
  • the embodiments of this method do preferably not include diagnostic methods practiced on the human or animal body.
  • subjects that are diagnosed to be at risk for developing a cardiometabolic disease and put on a therapy can be monitored, recorded and, if necessary (the selection step (b) also encompasses not changing the therapy, i.e. maintaining the applied therapy), their therapy can subsequently be adjusted or changed, corresponding to the selection step (b), depending on whether further risk factors of a cardiometabolic disease are determined, and to which extent, or whether further risk factors of a cardiometabolic disease are not determined.
  • the recorded data provides the basis for the assessment whether the therapy applied is beneficial to the patient or not.
  • step (a') a further step (b') of monitoring the therapy selected in step (b) is performed.
  • the invention relates to a kit comprising or consisting of one or more peptide sequence(s) of mannan-binding lectin serine peptidase 1 (MASP-1 ), glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1), thrombospondin (THBS1) and/or apolipoprotein A-IV (APOA-IV), wherein said peptide sequence(s) of MASP-1 is/are selected from TGVITSPDFPNPYPK, AAGNECPELQPPVHGK and SLPTCLPVCGLPK, wherein said peptide sequence(s) of GPLD1 is/are selected from IADVTSGLIGGEDGR, AQYVLISPEASSR and FGSSLITVR, wherein said peptide sequence of THBS1 is GGVNDNFQGVLQNVR, and wherein said peptide sequence(s) of APOA-IV is/are selected from SELTQQLNALF
  • This embodiment relates to a kit that comprises or consists of one or more of the above specified peptide sequences.
  • These peptide sequences i.e. peptide molecules that consist of the indicated peptide sequence, can be used as internal peptide standards in the quantification of the proteins described herein to be markers for the predisposition to develop T2D using a mass spectrometry based method such as, e.g., preferably SRM-MS.
  • the peptide molecules can be present in the kit in labeled form, preferably with stable isotope-labeled standards as defined in the respective passage above.
  • the isotopes employed are stable isotopes of carbon, nitrogen or hydrogen, such as e.g.
  • Labeling in this regard means that the N-terminal amino acid of the peptide sequence has said stable isotopes of carbon, nitrogen or hydrogen incorporated.
  • kits may comprise or consist of peptide sequences, preferably labeled, TGVITSPDFPNPYPK, AAGNECPELQPPVHGK and SLPTCLPVCGLPK; IADVTSGLIGGEDGR, AQYVLISPEASSR and FGSSLITVR; GGVNDNFQGVLQNVR; and/or SELTQQLNALFQDK, LGEVNTYAGDLQK and ISASAEELR.
  • TGVITSPDFPNPYPK AAGNECPELQPPVHGK and SLPTCLPVCGLPK
  • IADVTSGLIGGEDGR AQYVLISPEASSR and FGSSLITVR
  • GGVNDNFQGVLQNVR GGVNDNFQGVLQNVR
  • SELTQQLNALFQDK LGEVNTYAGDLQK and ISASAEELR.
  • the kit can comprise or consist of the peptide sequences of MASP-1 and GPLD1 , MASP-1 and THBS1 , MASP-1 and APOA-IV, GPLD1 and THBS1 , GPLD1 and APOA-IV, THBS1 and APOA-IV, MASP-1, GPLD1 and THBS1 , MASP-1 , GPLD1 and APOA-IV, MASP-1 , THBS1 and APOA-IV, GPLD1 , THBS1 and APOA-IV, or MASP-1 , GPLD1, THBS1 and APOA-IV.
  • the kit comprises or consists of peptide sequences of the additional proteins, preferably in labeled form, wherein the peptide sequences for APOE are selected from SELEEQLTPVAEETR, AATVGSLAGQPLQER and LEEQAQQIR; wherein the peptide sequences for CLEC3B are selected from LDTLAQEVALLK and EQQALQTVCLK; wherein the peptide sequences for CRP are selected from ESDTSYVSLK and GYSIFSYATK; wherein the peptide sequences for GSN are selected from EVQGFESATFLGYFK, AGALNSNDAFVLK and TPSAAYLWVGTG AS EAE K; wherein the peptide sequences for MBL2 are selected from WLTFSLGK, FQASVATPR and NAAENGAIQNLIK; wherein the peptide sequences for L
  • the peptide sequences of the additional proteins are part of the kit in combinations that are accordance with the protein combinations mentioned herein above in relation to the method of identifying a predisposition for developing a cardiometabolic disease in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease.
  • the kit comprises or consists of all peptide sequences of the proteins of the combination MASP-1 , GPLD1, THBS1 , APOA-IV, CD5L, GSN, ITIH4, APOE, CLEC3B, CRP, LBP, MBL2 and PZP.
  • the kit further comprises or consists of a sample preparation kit comprising a reagent and enzyme mixture for protein digestion of the sample/cell to be used in the respective methods of the invention using mass spectrometry analysis as described herein, optionally, also a standard protein to control for efficiency of reaction.
  • the kit preferably further comprises or consists of information for the mass spectrometry what peptides sequences or peptide sequence fragments are to be measured. This information is required to reliably measure the targeted peptides in targeted mass spectrometry, such as SRM-MS.
  • the information can be part of the kit in the form of a data carrier, e.g., with an executable code, which when provided to the control unit of the mass spectrometer provides the mass spectrometer with instructions as to what to measure.
  • the information may be part of a soft copy.
  • the information to be used in the case of SRM-MS can, e.g., be the information contained in the following table.
  • MS mass spectrometer
  • tryptic peptides are at least doubly charged in solutions used for electro spray ionization (ESI) mass spectrometry.
  • ESI electro spray ionization
  • the kit can preferably further comprise or consist of controls for retention times for applicability of "scheduled measurements" of peptides and peptide fragments.
  • kits may be packaged in one or more containers such as one or more vials.
  • the kit preferably further comprises preservatives or buffers for storage.
  • the kit may contain instructions for use.
  • the present invention further relates to the use of the kit of the invention in a method of identifying a predisposition for developing a cardiometabolic disease in a subject, or for producing a diagnostically informative value of the amount of one or more protein(s) or mRNA(s) encoding said protein(s) for identifying a predisposition for developing a cardiometabolic disease and/or in a method of identifying a compound capable of preventing a cardiometabolic disease and diseases associated therewith or serving as a lead compound for developing a compound capable of preventing a cardiometabolic disease and diseases associated therewith in accordance with the present invention.
  • the present invention further relates to the use of the kit of the invention as standard in the quantification of the proteins as defined in accordance with said kit.
  • quantification is by mass spectrometry.
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from.
  • a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I
  • the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C,
  • the KORA F4 study is a follow-up of the KORA S4 study, a population-based health survey conducted in the city of Augsburg and two surrounding counties between 1999 and 2001. A total sample of 6640 subjects was drawn from the target population consisting of all German residents of the region aged 25 to 74 years (Huth C, Beuerie S, Zierer A et al (2015) Biomarkers of iron metabolism are independently associated with impaired glucose metabolism and type 2 diabetes: the KORA F4 study. Eur J Endocrinol 173:643-65).
  • MASP1 , THBS1 , GPLD1 and ApoA- IV are novel biomarkers associated with prediabetes: the KORA F4 study.
  • the following text is taken from this publication: We performed a cross-sectional analysis of participants in the population-based KORA F4 study.
  • the plasma samples from 250 male and 250 female KORA F4 participants were randomly selected for SRM-MS from 1705 individuals fulfilling the following criteria: (1 ) omics data were available, to allow extended analyses in future studies; (2) within the age range of 47-76 years in which most of the KORA F4 omics data were available; (3) BMI data were available; (4) at least nine frozen plasma straws were available; and (5) individuals were either non-diabetic with fasting glucose, 2 h glucose and fasting insulin data, or had type 2 diabetes.
  • the 500 samples were randomly distributed into six sample preparation batches. The complete SRM-MS workflow was blinded to disease status. The analysis order was randomized.
  • One additional individual was excluded because of missing data in the C-reactive protein (CRP) covariate, leaving data from 439 participants for analysis of the dichotomous responses: prediabetes and type 2 diabetes vs normal glucose tolerance.
  • CRP C-reactive protein
  • the analyses of continuous fasting glucose, fasting insulin and insulin resistance were restricted to 406 participants who were not taking glucose-lowering medication. Two hour glucose levels were investigated in only 398 participants because there were missing values for eight participants who did not receive OGTTs because they were known to have diabetes (data not shown).
  • Non-SRM-MS measurements and variable transformations in KORA F4 All KORA F4 participants underwent a standard physical and medical examination. Participants classified as having known diabetes included all participants with self-reported diabetes that was validated by contacting the responsible physician or medical chart review, and those self-reporting a current intake of glucose-lowering medication. Additionally, a validation of the diabetes type was requested. If no validation but also no contradicting information was given, participants were assumed to have T2D. All participants without known diabetes of any type underwent a standard 75 g OGTT. Blood samples were taken without stasis after an overnight fast of at least 8 h and 2 h after glucose solution intake.
  • Circulating glucose was analyzed using a hexokinase method (GLU Flex, Dade Behring, Deerfield, IL, USA).
  • Normal glucose tolerance, prediabetes, and newly diagnosed diabetes were defined according to the 2003 ADA and according to the 1999 WHO criteria.
  • Known diabetic participants for whom the diabetes type could not be validated and for whom no contradictory information was given and newly OGTT-diagnosed diabetic cases were assumed to have type 2 diabetes.
  • Information about age, sex, smoking, alcohol intake and physical activity were assessed during a standardized interview, carried out by trained medical staff. Regular smoking was defined as smoking of at least one cigarette per day on average.
  • Alcohol intake was categorized as no (0 g/day), moderate (men 0.1-39.9 g/day; women 0.1-19.9 g/day) and high (men >40 g/day; women >20 g/day).
  • Individuals who did not participate in leisure time physical activity for at least one hour per week during both summer and winter were classified as physically inactive.
  • Actual hypertension was defined as systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mm Hg and/or use of antihypertensive medication given that the subjects were aware that they had hypertension.
  • Apolipoprotein A-IV was determined from frozen plasma using a double-sandwich ELISA (on an EVO150 TECAN pipetting robot) (Stangl et al., Journal of internal medicine, 278, 410-423, 2015).
  • the Skyline software was used to create SRM assays for 24 proteins including 1 control protein (MacLean et al., Bioinformatics. 2010 Apr 1 ;26(7):966-8) using the proposed workflow of empirical refinement (Bereman et al., Proteomics. 2012 Apr; 12(8): 1134-41.
  • Prior acquired shotgun peptide data were used to empirically select transitions as described before (von Toerne et al., J Proteome Res. 2013 Mar 1 ;12(3):1331-43) with the following changes: peptides selected from Progenesis LC-MS peptide result list, a Mascot Percolator score above 5.
  • Skyline peptide settings were as follows: Trypsin cutting [KR
  • ESI-LC-SRM-MS analysis for the targeted approach was performed on a TempoTM nano MDLC system (Eksigent) coupled online to a QTrap4000 (AB SCIEX) mass spectrometer by a nano spray ion source.
  • a TempoTM nano MDLC system Eksigent
  • QTrap4000 AB SCIEX
  • the digested samples were diluted five-fold in 2 % acetonitrile/0.5 % TFA and centrifuged for 5 min at 4°C before loading.
  • Peptides were trapped on a nano trap column (300 pm inner diameter ⁇ 5 mm, packed with Acclaim PepMaplOO C18, 5 pm, 100 A; LC Packings) in 0.1 % TFA at a flow rate of 20 ⁇ /min for 5 min and separated on an analytical column (75 pm inner diameter ⁇ 15 cm, Acclaim PepMap C18, 3 pm, 100 A, nanoViper; LC Packings) by a 90 min non-linear gradient using 2 % acetonitrile and 3 % DMSO and 0.1 % formic acid in water (A) and 95 % acetonitrile and 3% DMSO and 0.1 % formic acid in (B) at a flow rate of 250 nl/min.
  • the gradient settings were: 5-65 min: 3-40 % B, 65-75 min: 40-90 % B, 75-77 min: 90 % B, 79-80 min: 90-5 % B, followed by re-equilibration for 10 min to starting conditions.
  • the MS was used in positive ion mode with 2600 V ion spray voltage, curtain gas setting of 14, ion source gas setting of 30, nebulizer gas setting of 0, and an interface heater temperature of 170 °C.
  • Eluted peptides were measured with a scheduled SRM method in a SRM detection window of 360 sec and a target scan time of 3 sec.
  • the resolution in Q1 und Q3 was set to unit (0.7 amu FWHM).
  • RQ-SRM data generated from 500 samples were loaded into the Skyline software and displayed as first derivative transformation.
  • the here used mass spectrometer measured peptides, so-called precursor ions and multiple collision induced dissociation products of peptides, so-called fragment ions or transitions.
  • Isotope labeled, synthetic peptides (heavy peptides, PEPotecTM Thermo Fisher Scientific GmbH, Ulm, Germany) are used as internal controls of correct signal integration and for relative quantification. Relative quantification was based on the ratios of transitions of the endogenous peptide (light) compared to transitions of the respective heavy peptide (heavy).
  • AUC area under the curve
  • the preprocessing step is necessary to ensure the stability of the quantification relevant information and to condense the data.
  • First step of preprocessing was the exclusion of transitions without measurements for both, the heavy and the corresponding light isotope. Then, the coefficient of variation (CV) was calculated based on five replicates of a compound of all samples using the software AuDIT (Abbatiello et al., Clin Chem. 2010 Feb;56(2):291-305) and transitions with a CV of > 30% were excluded. Light and heavy transitions equaling zero (in total 0.25% of all data values) were replaced with a randomly drawn number between 1 and the minimum observation of the regarding transition. By doing this, no values below the limit of quantification (LOQ) threshold had to be deleted.
  • LOQ limit of quantification
  • the quantitative information was compressed to 2 transitions per peptide. Signals of transitions were compared and ranked by similarity. The two most similar signals were assumed to give the most reliable quantitation and were used for further calculations: (a) Within each peptide and separately for light and heavy isotope, the ratio of all transition combinations was calculated, yielding light relative ratio (LRR) and heavy relative ratios (HRR) for all transition combinations within each peptide, (b) Then the ratio of LRR and HRR for each transition combination was calculated, yielding the light to heavy relative ratio (LHRR) per transition combination. Good quality measurements should have a LHRR of approximately 1. (c) The average LHRR of all subjects for each transition combination was calculated.
  • LHR light to heavy ratios
  • model 1 was adjusted for technical covariates, age and sex; model 2 was adjusted for model 1 covariates and additionally for BMI; model 2b was adjusted for model 2 covariates and additionally for smoking status, alcohol intake, physical inactivity, and actual hypertension; model 3 was adjusted for model 2 covariates and additionally for smoking status, alcohol intake, physical inactivity, actual hypertension, Iog10-transformed triacylglycerol levels and Iog10-transformed total cholesterol/HDL- cholesterol ratio. In model 3 all proteins except CRP were additionally adjusted for log 10- transformed hsCRP, as measured by latex-enhanced immunonephelometry.
  • KORA F4 participants of whom plasma protein concentrations were measured in lot 2) A previously established set of 23 plasma proteins (for list see headers of Tables 8 and 9) was measured in samples from 600 additional KORA F4 study participants. The eligibility criteria for these additional measurements were: 1) between 42 and 81 years old at KORA F4, 2) no prevalent type 2 diabetes mellitus (T2D) at KORA F4, 3) valid 75-g oral glucose tolerance test (OGTT) data at KORA F4, 4) participation in the follow-up study KORA FF4 (2013/14), 5) either valid 75 g OGTT data at KORA FF4 or development of incident T2D between KORA F4 and KORA FF4.
  • T2D type 2 diabetes mellitus
  • OGTT 75-g oral glucose tolerance test
  • 600 plasma samples were selected according to the following criteria: 1 ) a random subsample of 353 participants, 66 additional incident type 2 diabetic participants, 181 additional incident prediabetic subjects according to ADA 2003 definition (Diabetes care, 26 Suppl 1, S5-20, 2003).
  • KORA FF4 study (data from this follow-up study was only used in the combined lot 1 and lot 2 analyses)
  • the KORA FF4 study is the second follow-up of the KORA S4 study.
  • 2279 also participated in the 14-year follow-up FF4 study.
  • KORA F4 and KORA FF4 were carried out in accordance with the Declaration of Helsinki, including written informed consent of all participants. All study methods were approved by the ethics committee of the Bavarian Chamber of Physicians, Kunststoff (EC No. 06068). Combination of measurement lots 1 and 2 in KORA F4 participants, and response definitions
  • FPG fasting plasma glucose
  • 2-h PG plasma glucose 2 hours post glucose-load during OGTT
  • prediabetes is the combination of i-IFG, i-IGT and IFG+IGT
  • the sample size for the investigation of incident T2D or prediabetes according to ADA-definition was: 328 incident prediabetic or type 2 diabetic versus 268 in KORA FF4 still normal glucose tolerant subjects.
  • the sample size for the investigation of incident T2D or prediabetes according to WHO-definition was: 204 incident prediabetic or type 2 diabetic versus 385 in KORA FF4 still normal glucose tolerant subjects.
  • Quantification was performed identical for lot 2 as for previous lot 1 measurements.
  • the data preprocessing procedure was performed using R version 3.2.2 (R Core Team, 2014, A Language and Environment for Statistical Computing, R Foundation for Statistical Computing). Preprocessing was done separately for data from lot 1 and lot 2 and was repeated for lot 1 in order to combine data from lot 1 and lot 2 which was preprocessed as similar as possible. Two transitions, which could not be measured in lot 2, were excluded in the new data preprocessing of lot 1.
  • Transitions with a CV of >30% were excluded (lot 1 : 5 transitions; lot 2: 33 transitions). Transition measurements that were zero (lot 1 : 0.25% and lot 2: 0.05% of all data values in the respective lot) were imputed by randomly assigning a number between 1 and the minimum observation of the respective transition. For each peptide with three transitions, the two transitions with the most similar information were selected for use in further calculations (an adaptation of a method established by Mani et al in 2012) ⁇ Mani et al., BMC bioinformatics, 13 Suppl 16, S9, 2012).
  • model 1 for age and sex
  • model 2 for model 1 covariates and additionally for BMI
  • model 3 for model 2 covariates and additionally for smoking status, alcohol intake, physical inactivity, actual hypertension, Iog10-transformed triacylglycerol levels and Iog10-transformed total cholesterol/HDL-cholesterol ratio.
  • model 3 all proteins except CRP were additionally adjusted for Iog10-transformed hsCRP, as measured by latex-enhanced immunonephelometry.
  • stepwise (back- and forward) model selection was applied including the LHRs of all 23 proteins at once while fixing the covariates of the respective adjustment model (for all three adjustment models).
  • the Akaike Information Criterion (AIC) was used as model selection criterion.
  • the model performance was assessed by calculating the AUC estimate of the base model (including only covariates adjusted for in the different adjustment models) and of the extended model (additionally including the by stepwise model selection selected protein-level LHRs). A p value of ⁇ 0.05 was considered statistically significant.

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Abstract

La présente invention concerne un procédé pour identifier une prédisposition au développement du diabète sucré de type 2 chez un sujet, ou pour produire une valeur à visée d'information diagnostique concernant la quantité d'une ou de plusieurs protéines ou d'un ou de plusieurs ARNm codant ladite ou lesdites protéines pour identifier une prédisposition au développement d'un diabète sucré de type 2, ledit procédé comprenant ou étant constitué de (i) l'évaluation dans un échantillon obtenu auprès dudit sujet de la quantité d'un ou de plusieurs protéines et/ou d'un ou de plusieurs ARNm codant ladite ou lesdites protéines, ladite ou lesdites protéines étant choisies dans un groupe de protéines défini dans ce qui suit, et (ii) la détermination des paramètres dudit sujet, dans lequel (a) lesdits paramètres sont l'âge et le sexe, et ledit groupe est constitué de AP0C2, AP0C3, APOE, CRP, MASP-1, ADIPOQ, ΙΤΊΗ4 et SHBG; (b) lesdits paramètres sont l'âge, le sexe et l'IMC, et ledit groupe est constitué de AP0C2, AP0C3, APOE, MASP-1, ADIPOQ, ITIH4, PZP et SHBG; ou (c) lesdits paramètres sont l'âge, le sexe, l'IMC, le statut de tabagisme, la consommation d'alcool, l'inactivité physique, l'hypertension réelle, les teneurs en triacylglycérol transformées en log10 et le rapport cholestérol total/HDL-cholestérol transformé en log10 et ledit groupe est constitué de MASP-1, ADIPOQ, ITIH4, ORM1 et PZP; dans lequel une augmentation de la quantité d'APOC2, APOC3, APOE, de CRP ou de MASP-1 ou d'un ARNm codant APOC2, APOC3, APOE, CRP ou MASP-1 ou une diminution de la quantité d'ADIPOQ, ITIH4, SHBG, PZP ou ORM1 ou d'un ARNm codant ADIPOQ, ITIH4, SHBG, PZP ou ORM1 par rapport à la quantité de ladite ou desdites protéines ou dudit ou desdits ARNm, respectivement, d'un témoin, indique une prédisposition à développer un diabète sucré de type 2.
PCT/EP2016/070011 2015-08-24 2016-08-24 Biomarqueurs pour maladies cardiométaboliques Ceased WO2017032815A2 (fr)

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Cited By (4)

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CN107024590A (zh) * 2017-03-13 2017-08-08 新疆医科大学 一种诊断1型和2型糖尿病的血清蛋白标志物组
WO2021142200A1 (fr) * 2020-01-10 2021-07-15 Somalogic, Inc. Méthodes de détermination d'une intolérance au glucose
KR20210099207A (ko) * 2016-07-21 2021-08-11 클리블랜드 하트랩, 아이엔씨. Hdl-관련 단백질 바이오마커 패널 검출
JP2022512602A (ja) * 2018-10-04 2022-02-07 コリア アドヴァンスド インスティテュート オブ サイエンス アンド テクノロジー 活性成分としてｔｓｐ１タンパク質インヒビターを含有する、ファブリー病を予防または処置するための医薬組成物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009064901A2 (fr) * 2007-11-13 2009-05-22 Veridex, Llc Biomarqueurs diagnostiques du diabète

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210099207A (ko) * 2016-07-21 2021-08-11 클리블랜드 하트랩, 아이엔씨. Hdl-관련 단백질 바이오마커 패널 검출
KR102424550B1 (ko) 2016-07-21 2022-07-22 클리블랜드 하트랩, 아이엔씨. Hdl-관련 단백질 바이오마커 패널 검출
CN107024590A (zh) * 2017-03-13 2017-08-08 新疆医科大学 一种诊断1型和2型糖尿病的血清蛋白标志物组
JP2022512602A (ja) * 2018-10-04 2022-02-07 コリア アドヴァンスド インスティテュート オブ サイエンス アンド テクノロジー 活性成分としてｔｓｐ１タンパク質インヒビターを含有する、ファブリー病を予防または処置するための医薬組成物
JP2023093504A (ja) * 2018-10-04 2023-07-04 コリア アドヴァンスド インスティテュート オブ サイエンス アンド テクノロジー 活性成分としてｔｓｐ１タンパク質インヒビターを含有する、ファブリー病を予防または処置するための医薬組成物
WO2021142200A1 (fr) * 2020-01-10 2021-07-15 Somalogic, Inc. Méthodes de détermination d'une intolérance au glucose
JP2023509677A (ja) * 2020-01-10 2023-03-09 ソマロジック オペレーティング カンパニー インコーポレイテッド 耐糖能異常障害を決定する方法

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