WO2011029201A1

WO2011029201A1 - Methods and kits for the detection of heart disease

Info

Publication number: WO2011029201A1
Application number: PCT/CA2010/001426
Authority: WO
Inventors: Sara Arab; Peter Liu; Mark Suchul Moon; Liyong Zhang
Original assignee: University Health Network; University of Toronto
Current assignee: University Health Network; University of Toronto
Priority date: 2009-09-11
Filing date: 2010-09-13
Publication date: 2011-03-17
Anticipated expiration: 2012-03-11

Abstract

The disclosure provides biomarkers for detecting heart disease, including biomarkers detectable in blood. The disclosure provides a method of screening for, diagnosing or detecting heart disease in a subject, the method comprising, a) determining a level of Mindin in the sample, and b) comparing the level of Mindin in the sample with a control, wherein an increased level of Mindin in the sample compared to the control is indicative that the subject has heart disease.

Description

Title: Methods and Kits for the Detection of Heart Disease

[0001] This Patent Cooperation Treaty application claims the benefit of 35 USC 19 based on the priority of co-pending US Provisional Patent Application 61/241 ,648, filed September 1 1 , 2009 which is incorporated herein in its entirety by reference.

Field of the Disclosure

[0002] The disclosure relates to methods and compositions for the detection of heart disease and specifically to the use of biomarkers and compositions comprising agents that bind the biomarkers for the detection of heart disease. Background of the Disclosure

[0003] Cardiovascular diseases are the most important cause of morbidity and mortality in developed countries, causing twice as many deaths as cancer in the USA. The need for diagnostic and prognostic biomarker and new targets for therapy is evident. Mindin

[0004] Mindin (Spondin 2) is a highly conserved secreted extracellular matrix (ECM) protein of the Mindin-F-spondin family. Previous studies showed Mindin to be a regulator of host innate immunity, but despite its high expression in the heart, its role in cardiac stress response is unknown. HACE1

[0005] Hace is a HECT family E3 ubiquitin-protein ligase gene, HACE1 , and a potential suppressor of multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late onset tumors (Zhang, 2007).

Summary of the Disclosure

[0006] Biomarkers useful for diagnosing heart disease are disclosed herein.

Treatments targeting the biomarkers are useful for treating heart disease.

[0007] It is disclosed herein that mice that have a loss of function for Mindin (Mindin -/-mice) show lower mortality and morbidity post myocardial infarction. Minden expression is associated with higher mortality and acute adverse remodelling post myocardial infarction. Further it is demonstrated herein that Minden levels are increased in human serum of ischemic subjects compared to controls.

[0008] Accordingly, in an aspect the disclosure provides a method of screening for, diagnosing or detecting heart disease in a subject, the method comprising: a. determining a level of Mindin in a sample of the subject; and b. comparing the level of Mindin in the sample with a control; wherein an increased level of Mindin in the sample compared to the control is indicative that the subject has heart disease.

[0009] In another embodiment, the method comprises: a) obtaining a sample from the subject;

b) determining a level of Mindin in the sample, and

c) comparing the level of Mindin in the sample with a control; wherein an increased level of Mindin in the sample compared to the control is indicative that the subject has heart disease.

[0010] In another aspect the disclosure provides a method for treating a subject having heart disease comprising:

a. determining a level of Mindin in a sample from the subject, and b. treating the subject with a treatment for heart disease when the level of Mindin in the sample is increased compared to control.

[001 1] Yet a further aspect provides a method of monitoring response to treatment comprising: a. determining a base-line level of Mindin in a base-line sample from the subject;

b. determining a level of Mindin in a post-treatment sample from the subject; and

c. comparing the level of Mindin in the post-treatment sample with the base-line level; wherein an increase in the level of Mindin in the post-treatment sample compared to the baseline level is indicative the subject is not responding or is responding poorly to treatment, and a decrease in the level of Mindin in the post treatment sample compared to the base-line level is indicative that the subject is responding to treatment.

[0012] Another aspect provides a method of monitoring heart disease progression comprising: a. determining a base-line level of Mindin in a base-line sample from the subject;

b. determining a level of Mindin in a sample taken subsequent to the base-line sample from the subject; and

c. comparing the level of each biomarker in the sample with the base-line level;

wherein an increase in the Mindin level in the post-base-line sample compared to the base-line level is indicative the heart disease is progressing, and a decrease in the biomarker level in the post base-line sample compared to the baseline level is indicative that the heart disease is not progressing.

[0013] In an embodiment, the heart disease is ischemia. In another embodiment the heart disease dilated cardiomyopathy.

[0014] In an embodiment, the level of Mindin is determined using a biomarker specific reagent, such an antibody or antibody fragment, which specifically binds Mindin.

[0015] It is also demonstrated herein that Hacel is useful for differentiating systolic heart failure from diastolic heart failure. Hacel gene and protein expression levels are significantly elevated in blood samples of systolic heart failure patients. Further Hacel knock out mice fail to develop hypertrophy following cardiac banding.

[0016] Accordingly, in an aspect the disclosure provides a method of screening for, diagnosing or detecting heart disease in a subject, the method comprising: a. determining a level of Hacel in a sample from the subject; and b. comparing the level of Hacel in the sample with a control; wherein an increased level of Hacel in the sample compared to the control is indicative that the subject has heart disease.

[0017] In another aspect the disclosure provides a method for treating a subject having heart disease comprising:

c. determining a level of Hacel in a sample from the subject, and d. treating the subject with a treatment for heart disease when the level of Hacel in the sample is increased compared to control.

[0018] Yet a further aspect provides a method of monitoring response to treatment comprising:

a. determining a base-line level of Hacel in a base-line sample from the subject;

b. determining a level of Hacel in a post-treatment sample from the subject; and

c. comparing the level of Hacel in the post-treatment sample with the base-line level; wherein an increase in the level of Hacel in the post-treatment sample compared to the baseline level is indicative the subject is not responding or is responding poorly to treatment, and a decrease in the level of Hacel in the post treatment sample compared to the base-line level is indicative that the subject is responding to treatment.

[0019] Another aspect provides a method of monitoring heart disease progression comprising: a. determining a base-line level of Hacel in a base-line sample from the subject; b. determining a level of Hacel in a sample taken subsequent to the base-line sample from the subject; and

wherein an increase in the Hacel level in the post-base-line sample compared to the base-line level is indicative the heart disease is progressing, and a decrease in the biomarker level in the post base-line sample compared to the baseline level is indicative that the heart disease is not progressing.

[0020] In an embodiment, the heart disease is systolic heart failure.

[0021] In a further aspect, the disclosure provides a method of distinguishing systolic heart failure from diastolic heart failure in a subject with heart failure, the method comprising:

a. determining a level of Hacel in a sample from the subject, and b. comparing the level of Hacel in the sample with a control, wherein the control is a level of Hacel in a subject without heart failure or a subject with diastolic heart failure;

wherein an increased level of Hacel in the sample compared to the control is indicative the subject has systolic heart failure and wherein a level comparable in the sample to the control is indicative the subject has diastolic heart failure.

[0022] In an embodiment, the level of Hacel is determined using a biomarker specific reagent, such an antibody or antibody fragment, which specifically binds Hacel .

[0023] In an embodiment, the sample and/or control comprises polypeptide. In an embodiment, the sample and/or control is a blood sample and comprises blood or a fraction thereof such as serum.

[0024] In an embodiment, the treatment is an agent that inhibits the biomarker such as an antibody specific for the biomarker or a siRNA molecule that inhibits the expression of the biomarker, for example injected in heart or administered intravenously. [0025] A further aspect provides a kit for detecting Mindin, and/or Hacel levels.

[0026] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

[0027] An embodiment of the present disclosure will now be described in relation to the drawings in which:

Figure 1. Western blots shows Mindin expression in tissues and post Ml

Figure 2. Western blot and graphs show Mindin expression in human serum fro control and ischemic subjects.

Figure 3. Western blots shows HACE1 protein levels is elevated in blood samples of systolic heart failure patient but not in blood samples from diastolic heart failure patient. DHF-diastolic heart failure; SHF-systolic heart failure.

Figure 4. Human heart tissue microarray showing Mindin imRNA expression.

Figure 5. Western blott and graph showing Mindin expression ICM and DCM human serum.

Detailed description of the Disclosure

[0028] In the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. I. Definitions

[0029] The phrase "screening for, diagnosing or detecting ischemia" refers to a method or process of determining if a subject has or does not have heart disease.

[0030] The term "subject" as used herein refers to any member of the animal kingdom, preferably a human being including for example a subject that has or is suspected of having heart disease such as heart failure.

[0031 ] The term "level" as used herein refers to an amount or quantity (e.g. relative amount or concentration) of biomarker that is detectable or measurable in a sample. For example, the level can be a concentration such as pg/L or a relative amount such as 1.1 , 1.2, 1.3, 1.4, 1 .5, 1.6, 1.7, 1 .8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 10, 15, 20, 25, 30, 40, 60, 80 and/or 100 times a control level, where for example, the control level is the level such as the average or median level in a normal sample (e.g. serum from a subject without heart failure). In an embodiment, where the sample is a blood sample, the level is the level of polypeptide. In another embodiment, the level is the level of mRNA or cDNA. [0032] The term "determining a level" as used in reference to a biomarker means the application of a method to a sample, for example a sample of the subject and/or a control sample, for ascertaining quantitatively, semi-quantitatively or qualitatively the amount of a biomarker, for example the amount of biomarker polypeptide or mRNA. For example, a level of a biomarker can be determined by a number of methods including for example mass spectrometric methods, including for example MS, MS/MS, LC- MS/MS, SRM etc where a peptide of a biomarker is labeled and the amount of labeled biomarker peptide is ascertained, immunoassays including for example immunohistochemistry, Western blotting, ELISA, immunoprecipitation and the like, where a biomarker biomarker specific reagent such as an antibody for example, a labeled antibody specifically binds the biomarker and permits for example relative or absolute ascertaining of the amount of polypeptide biomarker, hybridization and PCR protocols, where a probe or primer or primer set are used to ascertain the amount of nucleic acid biomarker, including for example RT-PCR. [0033] The term "increase in the level" as used herein refers to an increase in the level or quantity of a biomarker described herein, for example Mindin or Hacel , in a sample from the subject that is measurable compared to a suitable control and/or reference. Similarly the term "decrease in the level" as used herein refers to a decrease in the level or quantity of a biomarker described herein, for example Mindin or Hacel , in a sample from the subject that is measurable compared to a suitable control and/or reference For example, the increase or decrease can be an increase or decrease in the steady-state level of a gene transcript, including for example a difference resulting from a difference in the level of transcription and/or translation and/or transcript degradation. For example, the increase or decrease in the level can refer to an increase or decrease in the measurable polypeptide or fragment thereof level of a given biomarker as measured by the amount of steady state level of and/or expressed polypeptide or fragment thereof in a test sample as compared with the measurable expression level of a given biomarker or fragment thereof in a control, population of control samples and/or previously taken or reference sample, for example in a blood sample. In another example, the difference in the level can refer to an increase or decrease in the measurable nucleic acid transcript level of a given biomarker as measured by the amount of transcript e.g. biomarker mRNA or cDNA, for example as detectable in a sample comprising heart tissue. In methods relating to monitoring therapeutic response or disease progression, an increase or decrease in the level can refer to an increase or decrease in the level of the biomarker in the subsequent sample (e.g. post treatment sample) compared to a reference sample (e.g. baseline sample), wherein an increase is indicative of negative therapeutic response and/or disease progression and a decrease is indicative of a positive therapeutic response and/or disease stabilization or amelioration. For example, an increase or decrease in a level of biomarker level is detected if a ratio of the level in a test sample as compared with a control is greater than or less than 1 .0 and/or if the ratio of the level in a reference sample as compared with a subsequent sample is greater than or less than 1 .0. For example, a ratio of greater than 1.0, 1 .2, 1.5, 1.7, 2, 3, 3, 5, 10, 12, 15, 20 or more, or a ratio less than 1 , 0.8, 0.6, 0.4, 0.2, 0.1 , 0.05, 0.001 or less. [0034] The term "control" as used herein refers to a sample from an subject or a group of individuals who are known as not having heart disease or to a biomarker level or value reflective of individuals who are known as not having heart disease, at which or below which individuals are likely to not have heart disease or the particular subtype being determined by the method (e.g. diastolic heart failure). In addition, the control is optionally derived from tissue of the same type as the sample of the subject being tested. For example, the control can be a serum sample where the sample from the subject being tested is a serum sample. Where the control is a numerical value or range, the numerical value or range is a predetermined value or range that corresponds to a level of the biomarker or range of levels of the biomarker in a group of subjects known as not having heart disease (e.g. threshold or cutoff level; or control range) or a particular subtype of heart disease (e.g. ischemic heart disease). For example, the control can be a cut-off or threshold level, above or below which (depending on the biomarker and subtype) which a subject is identified as having heart disease or a particular type thereof. For example, a test subject that has an increased level of a biomarker above a cut-off or threshold level is indicated to have or is more likely to have heart disease or a particular type of heart disease.

[0035] The term "positive control" as used herein refers to a sample of an individual or a group of individuals with heart disease and/or a value e.g. corresponding to a level of one or more biomarkers associated with the disease class, e.g. systolic heart failure, cardiac ischemia, post-myocardial infarction etc. [0036] The term "baseline level" as used herein refers to a level that is used for comparison to a sample taken at a later time point. For example, in methods related to monitoring response to treatment or disease progression, "base-line level" can refer to a level of a biomarker in a sample taken prior to a subsequent sample, e.g. base line sample is taken before treatment, comparison to which provides an indication of response to treatment.

[0037] The term "biomarker" as used includes without limitation, a polypeptide or fragment, a polypeptide or fragment thereof that is secreted or released, for example which is secreted or released into blood (e.g. soluble biomarker), a nucleic acid sequence including a gene, or corresponding RNA, for example a RNA level in a heart tissue sample. The biomarkers of the disclosure include for example, Mindin, Hacel , their homologs and fragments thereof.

[0038] The term "polypeptide biomarker" refers to polypeptide and/or fragments thereof of a biomarker of the present disclosure and includes polypeptides translated from the RNA transcripts of biomarkers described herein. Polypeptide biomarkers include modified (e.g. post-translational modifications such as glycosylation), expressed, as well as soluble biomarkers such as secreted, cleaved, released, and shed polypeptide products. The terms "polypeptide" and "protein" are intended to be used interchangeably. [0039] The term "soluble biomarker" as used herein refers to a polypeptide biomarker, that is released in any manner from a cell and detectable in a blood sample, such as blood, serum, plasma.

[0040] The term "prognosis" as used herein refers to an expected clinical outcome group such as a poor survival group or a good survival group associated with or reflected by an increased biomarker level or levels when compared to a control, wherein the biomarker(s) is/are selected from the Mindin or Hacel or combinations thereof.

[0041] The term "sample" as used herein refers to any biological fluid, cell or tissue sample from a subject which can be assayed for biomarkers (e.g. RNA and/or polypeptide products), such as soluble biomarkers in subjects having or not having heart failure. For example the sample is optionally or comprises blood, serum, plasma, or heart cells. The sample can for example be a "post-treatment" sample wherein the sample is obtained after one or more treatments, or a "base-line sample" which is for example used as a base line for assessing disease progression. In a preferred embodiment, the sample is a blood sample.

[0042] The term "blood sample" as used herein refers to any fraction or whole blood, which can comprise cells or be substantially cell free, which can be assayed for biomarkers, including for example blood, serum, and plasma. [0043] The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds- scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0044] Antibodies having specificity for a specific protein, such as the protein product of a biomarker of the disclosure, may be prepared by conventional methods. A mammal, (e.g. a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera. [0045] To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g. the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495- 497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4:72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Methods Enzymol, 121 :140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.

[0046] In aspects, the antibody is a purified or isolated antibody. By "purified" or "isolated" is meant when for example referring to an antibody, that a given antibody or fragment thereof, whether one that has been removed from nature (isolated from blood serum) or synthesized (produced by recombinant means), has been increased in purity, wherein "purity" is a relative term, not "absolute purity." In particular aspects, a purified antibody is 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated or associated following synthesis and/or during isolation. [0047] The term "biomarker specific reagent" as used herein refers to any molecule or compound that can bind to a biomarker product described herein, including polypeptides such as antibodies, aptamers, nucleic acids and peptide mimetics. The "biomarker specific reagent" can for example be coupled to or labeled with a detectable marker. The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ²³l, ¹²⁵l, ¹³¹l; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.

[0048] The term "hybridize" or "hybridizable" refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0 X sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 X SSC at 50° C may be employed.

[0049] The term "polynucleotide", "nucleic acid" and/or "oligonucleotide" as used herein refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, and is intended to include DNA and RNA which can be either double stranded or single stranded, represent the sense or antisense strand.

[0050] The term "primer" as used herein refers to a polynucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.

[0051 ] The term "probe" as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to a biomarker RNA or a nucleic acid sequence complementary to the biomarker RNA. The length of probe depends for example, on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. The probe can be for example, at least 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length. [0052] A person skilled in the art would recognize that "all or part of of a particular probe or primer can be used as long as the portion is sufficient for example in the case a probe, to specifically hybridize to the intended target and in the case of a primer, sufficient to prime amplification of the intended template.

[0053] The term "specifically binds" as used herein refers to a binding reaction that is determinative of the presence of the biomarker (e.g. polypeptide or nucleic acid) often in a heterogeneous population of macromolecules. For example, when the biomarker specific reagent is an antibody, specifically binds refers to the specified antibody binding with greater affinity to the cognate antigenic determinant than to another antigenic determinant, for example binds with at least 2, at least 3, at least 5, or at least 10 times greater specificity; and when a probe, specifically binds refers to the specified probe under hybridization conditions binds to a particular gene sequence at least 1.5, at least 2 at least 3, or at least 5 times background.

[0054] The term "kit control" as used herein means a suitable assay control useful when determining a level of a biomarker level For example, where the kit is for detecting polypeptide levels by immunohistochemical methods, the kit control can comprise an antibody control, useful for example for detecting non-specific binding and/or for standardizing the amount of protein in the sample. For example, the kit control can comprise an antibody that specifically binds vinculin.

[0055] The term "biomarker specific reagent" as used herein refers to a reagent that is a highly sensitive and specific biomarker reagent used with standard immunohistochemistry (ICC) and immunohistochemistry (IHC) techniques to detect the level of a biomarker associated with heart disease.

[0056] The term "Mindin", also known as DIL-1 , DILI , M-spondin and Spondin-2 precursor, refers to a Mindin gene (e.g. gene name SPON2 and DILI ) or expression products(e.g. mRNA and polypeptide products), or cDNA, including naturally occurring variants, and including mammalian Mindin, such as human Mindin, including those deposited in Genbank with accession number NM 012445, NM_001 128325, NP_001 121797, NP_036577, AAH36341 , AAH02707, 3D34_B, 3D34_A, BC002707, BC036341 , BD 88997, BD188996, AX576915, BD006593 and BD006592 and/or with UniProtKB/Swiss-Protien accession numbers Q9BUD6, Q4W5N4, Q9ULW1 (SPON2_HUMAN) and sequences included herein. Mindin is an extracellular matrix protein and the gene encoding Mindin is located on chromosome 4p16.3.

[0057] The term "Hacel" or "HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1 " refers to a Hacel gene or expression products (e.g. mRNA and polypeptide products) or cDNA including naturally occurring variants, and including mammalian Hacel , such as human Hacel , including those deposited in Genbank with accession number NP 065822, NM 020771 and sequences included herein. [0058] Sequences of accession numbers referred to herein are hereby incorporated by reference.

[0059] The term "heart disease" as used herein refers to a variety of disorders and diseases that affect the heart includes for example ischemic heart disease, cardiomyopathy including for example dilated cardiomyopathy (DCM), cardiovascular disease, heart failure, hypertrophy, and myocardial infarction induced injuries including initial injuries during the acute adverse remodeling phase.

[0060] The term "heart disease excluding myocardial infarction induced initial injuries" as used herein refers to a variety of disorders and diseases that affect the heart includes for example ischemic heart disease, cardiomyopathy including for example dilated cardiomyopathy (DCM), cardiovascular disease, heart failure, hypertrophy and myocardial infarction related injuries but not including initial injuries during the acute adverse remodeling phase.

[0061] The term "heart failure" as used herein refers to a to a clinical syndrome characterized by distinctive symptoms and signs resulting from disturbances in cardiac output or from increased venous pressure and includes chronic, acute and end-stage heart failure. Heart failure includes systolic heart failure and diastolic heart failure.

[0062] The term "ischemic heart disease", "ischemic cardiomyopathy", "ischemia" or cardiac ischemia as used herein refers to disorders characterized by reduced blood supply to the heart muscle.

[0063] The term "cardiac hypertrophy" as used herein refers to thickening and enlargement of the cardiac chamber, and includes established compensatory cardiac hypertrophy, and pathological decompensated cardiac hypertrophy.

[0064] The term "Dilated cardiomyopathy" or "DCM" as used herein refers to a type of cardiomyopathy in which the heart becomes weakened and enlarged, and left or right ventricular systolic pump function of the heart is impaired, leading to progressive cardiac enlargement and hypertrophy, in a process referred to as remodeling. Dilated cardiomyopathy is the most common form of non-ischemic cardiomyopathy. [0065] The term "myocardial infarction" or Ml ("heart attack") as used herein is the irreversible damage of myocardial tissue caused by for example, prolonged ischemia and hypoxia. Ml can result in permanent damage of the myocardium and increased morbidity as a consequence. The infarcted tissue typically forms a fibrotic scar. Long-term consequences include ventricular remodeling of the remaining myocardium (e.g., development of compensatory hypertrophy and/or dilation), ventricular failure, arrhythmias and sudden death. Improvement in a myocardial infarction induced injury can for example be determined by detecting improvement in systolic function (e.g. ejection fraction, fractional shortening) and improvement in diastolic function as result of inhibition of adverse cardiac remodeling (decreased infarct size, decreased replacement fibrosis and fewer dying cells), inhibition of hypertrophy, inhibition of chamber dilation, and consequently inhibition of ventricular fibrillation and arrhythmias, well as for example secondary pulmonary congestion and edema.

[0066] As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, reversal of disease, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0067] As used herein an "agent that inhibits a biomarker" refers to an agent that inhibits the expression or function of the biomarker and includes for example a neutralizing antibody specific for the biomarker that inhibits for example biomarker function or a siRNA molecule that inhibits the expression of the biomarker. The agents can, for example injected in heart.

[0068] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0069] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1 .5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise

II. Methods

[0070] It is disclosed herein that Mindin levels are increased in serum samples of subjects with ischemic heart disease and dilated cardiomyopathy. Mindin levels are increased in both ischemic and non-ischemic (e.g. dilated cardiomyopathy) heart disease, for example supporting that Mindin levels are increased in heart disease generally.

[0071 ] It is further disclosed herein that Hace-1 gene and protein expression levels are significantly elevated in the blood samples of systolic heart failure patients. Hace-1 knockout mice also fail to develop hypertrophy following cardiac banding, suggesting a role for Hace-1 in heart disease.

[0072] Accordingly, in an aspect the disclosure provides a method of screening for, diagnosing or detecting heart disease in a subject, the method comprising:

a. determining a level of Mindin, Hace-1 or a combination thereof in the sample, and

b. comparing the level of Mindin and/or Hace-1 in the sample with a control; wherein an increased level of Mindin and/or Hace-1 in the sample compared to the control is indicative that the subject has heart disease.

[0073] In an embodiment, the method comprises:

a. obtaining a sample from the subject;

b. determining a level of Mindin, Hace-1 or a combination thereof in the sample, and

c. comparing the level of Mindin and/or Hace-1 in the sample with a control; wherein an increased level of Mindin and/or Hace-1 in the sample compared to the control is indicative that the subject has heart disease.

[0074] In another aspect the disclosure provides a method for treating a subject having heart disease comprising:

a. determining a level of Mindin and/or Hace-1 in a sample from the subject, and

b. treating the subject with a treatment for heart disease when the level of Mindin and/or Hace-1 in the sample is increased compared to control.

[0075] In an embodiment, the method comprises: a. obtaining a sample from the subject;

b. determining a level of Mindin and/or Hace-1 in a sample from the subject, and

c. treating the subject with a treatment for heart disease when the level of Mindin and/or Hacel in the sample is increased compared to control.

[0076] Also provided is use of a heart disease treatment for treating subjects with an increased level of Mindin and/or Hacel compared to a control.

[0077] Yet a further aspect provides a method of monitoring response to treatment comprising:

a. determining a base-line level of Mindin and/or Hace-1 in a base-line sample from the subject;

b. determining a level of Mindin and/or Hace-1 in a post-treatment sample from the subject; and

c. comparing the level of Mindin and/or Hace-1 in the post-treatment sample with the base-line level; wherein an increase in the level of Mindin and/or Hace-1 in the post-treatment sample compared to the baseline level is indicative the subject is not responding or is responding poorly to treatment, and a decrease in the level of Mindin and/or Hace- 1 in the post treatment sample compared to the base-line level is indicative that the subject is responding to treatment.

[0078] Another aspect provides a method of monitoring heart disease progression comprising:

a. determining a base-line level of Mindin and/or Hacel in a base-line sample from the subject;

b. determining a level of Mindin and/or Hacel in a sample taken subsequent to the base-line sample from the subject; and

c. comparing the level of each biomarker in the sample with the baseline level;

wherein an increase in the Mindin and/or Hacel level in the post-base-line sample compared to the base-line level is indicative the heart disease is progressing, and a decrease in the Mindin and/or Hacel level in the post base-line sample compared to the base-line level is indicative that the heart disease is not progressing.

[0079] In an embodiment, the level of Mindin is determined. In an embodiment, the level of Hacel is determined.

[0080] In an embodiment, the Mindin level determined is a polypeptide level. In an embodiment, the Mindin polypeptide comprises SEQ ID NO:3 and/or 5 and/or a polypeptide with at least 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO:3 and/or 5. In an embodiment, the Mindin level determined is a nucleic acid level. In an embodiment, the Mindin nucleic acid comprises SEQ ID NO:1 and/or 2 and/or a nucleic acid molecule with at least 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO: 1 and/or 2.

[0081 ] In an embodiment, the Hace-1 level determined is a polypeptide level.

In an embodiment, the Hace-1 polypeptide comprises SEQ ID NO:6 and/or a polypeptide with at least 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO:6. In an embodiment, the Hace-1 level determined is a nucleic acid level. In an embodiment, the Hace-1 nucleic acid comprises SEQ ID NO:7 and/or a nucleic acid molecule with at least 95%, 96%, 97%, 98% or at least 99% sequence identity with SEQ ID NO: 7.

[0082] In an embodiment, the biomarker determined is soluble biomarker.

[0083] In an embodiment, the heart disease comprises ischemic heart disease. In another embodiment, the heart disease comprises a cardiomyopathy. In an embodiment, the cardiomyopathy comprises dilated cardiomyopathy (DCM). In an embodiment, the heart disease comprises cardiovascular disease. In another embodiment, the heart disease comprises heart failure. In yet another embodiment, the heart disease comprises hypertrophy. In an embodiment, the heart disease comprises injuries sustained resulting from a myocardial infarction. In a further embodiment, the heart disease is heart disease excluding myocardial infarction induced initial injuries.

[0084] In an embodiment, wherein the subject has an increased level of Mindin, the heart disease indicated comprises ischemic heart disease. In another embodiment, wherein the subject has an increased level of Mindin the heart disease indicated comprises myocardial infarction induced injury. In yet another embodiment, wherein the subject has an increased level of Mindin, the heart disease indicated comprises DCM.

[0085] In an embodiment, the method is for the early detection of heart disease. In an embodiment, the method is for the early detection of ischemic heart disease. As an example, it is demonstrated herein using an animal model, that Mindin shows elevation in the acute and early stage of disease (e.g. see Figure 1).

[0086]

[0087] In another embodiment, wherein the subject has an increased level of Hacel , the heart disease is systolic heart failure.

[0088] It is also demonstrated herein that Hacel is useful for differentiating systolic heart failure from diastolic heart failure. Further Hacel knock out mice fail to develop hypertrophy following cardiac banding. [0089] In a further aspect, the disclosure provides a method of distinguishing systolic heart failure from diastolic heart failure in a subject with heart failure, the method comprising: a. determining a level of Hacel in the sample, and

b. comparing the level of Hacel in the sample with a control, wherein the control is a level of Hacel in a subject without heart failure or a subject with diastolic heart failure;

[0090] In an embodiment, the method comprises: a. determining a level of Hacel in a sample from the subject, and b. comparing the level of Hacel in the sample with a control, wherein the control is a level of Hacel in a subject without heart failure or a subject with diastolic heart failure;

In an embodiment, the level of two biomarkers are determined.

[0091] In an embodiment, the method comprises obtaining a sample from the subject. The sample can for example be a blood sample or a biopsy.

[0092] In an embodiment, the ratio of the level of the biomarker in the sample compared to the control indicative of heart disease or a type thereof is greater than 1.2, 1.4, 1.6, 1 .8, 2, 3, 5, 10, or more. In an embodiment, the ratio is at least 1 .3, at least 1.5, at least 1.7, at least 1.9, at least 2.1 , at least 2.5 or at least 3.

[0093] In an embodiment, the level of the biomarker determined is a polypeptide level or a nucleic acid level.

[0094] In an embodiment, the level of Mindin is determined using a biomarker specific reagent, e.g a Mindin specific reagent such an antibody or antibody fragment, which specifically binds Mindin, for example Mindin polypeptide. In another embodiment, the Mindin specific reagent is a probe that hybridizes to Mindin mRNA or cDNA. In yet a further embodiment, the Mindin specific reagent is a primer or primer pair for amplifying Mindin nucleic acids. In another embodiment, the level of Hacel is determined using a biomarker specific reagent e.g. a Hacel specific reagent, such an antibody or antibody fragment, which specifically binds Hacel , for example Hacel polypeptide. In a further embodiment, the Hacel specific reagent is a probe that hybridizes to Hacel mRNA or cDNA. In yet a further embodiment, the Hacel specific reagent is a primer or primer pair for amplifying Hacel nucleic acids, e.g. for example by PCR methods.

[0095] A person skilled in the art will appreciate that a number of methods can be used to determine the amount of a polypeptide biomarker, including soluble biomarker of the disclosure, including mass spectrometry approaches, such as multiple reaction monitoring (MRM) and product-ion monitoring (PIM), and also including immunoassays such as Western blots, enzyme-linked immunosorbant assay (ELISA), and immunoprecipitation followed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immunocytochemistry. Accordingly, in other embodiments, the level determined is a polypeptide product. In certain embodiments, the step of determining the biomarker level comprises using immunohistochemistry and/or an immunoassay. In certain embodiments, the immunoassay is an ELISA. In yet a further embodiment, the ELISA is a sandwich type ELISA.

[0096] In an embodiment, antibodies or antibody fragments are used to determine the level of polypeptide of one or more biomarkers of the disclosure. In an embodiment the antibody or antibody fragment is labeled with a detectable marker. In a further embodiment, the antibody or antibody fragment is, or is derived from, a monoclonal antibody. A person skilled in the art will be familiar with the procedure for determining the level of a polypeptide biomarker by using said antibodies or antibody fragments, for example, by contacting the sample from the subject with an antibody or antibody fragment labeled with a detectable marker, wherein said antibody or antibody fragment forms a complex with the biomarker. [0097] The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³l, ¹²⁵l, ³ ; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.

In another embodiment, the level of polypeptide biomarker of the disclosure is detectable indirectly. For example, a secondary antibody that is specific for a primary antibody that is in turn specific for the isolated protein of the disclosure wherein the secondary antibody contains a detectable label can be used.

[0098] In an embodiment, the Mindin specific reagent specifically binds Mindin polypeptide. In an embodiment the Mindin specific reagent specifically binds SEQ ID NO:3 and/or 5. In an embodiment, the Mindin specific reagent specifically binds Mindin nucleic acid. In an embodiment, the Mindin specific reagent specifically binds SEQ ID NO: 1 and/or 2.

[0099] In an embodiment, the Hacel specific reagent specifically binds Hacel polypeptide. In an embodiment the Hacel specific reagent specifically binds SEQ ID NO:6. In an embodiment, the Hacel specific reagent specifically binds Hacel nucleic acid. In an embodiment, the Hacel specific reagent specifically binds SEQ ID NO:7.

[00100] In embodiments relating to treatment, the treatment is optionally an agent that inhibits the biomarker such as an antibody specific for the biomarker or a siRNA molecule that inhibits the expression of the biomarker, for example injected in heart or administered intravenously.

IV. Kits

[00101] Another aspect of the disclosure is a kit for screening for, detecting, or diagnosing heart disease in a subject and/or determining prognosis of a subject having heart disease. In an embodiment, the kit comprises one or more biomarker specific reagents, for example an antibody, specific for a biomarker described herein, for example Mindin or Hacel .

[00102] The kit can also include a control or reference standard e.g. such as a positive control such as Mindin or Hacel polypeptide and/or instructions for use. In addition, the kit can include ancillary agents such as vessels for storing or transporting the sample for example specimen collection tubes for example for collecting a biopsy, and/or storing or transporting biomarker specific reagents and/or buffers such as extraction buffers for extracting nucleic acid, for example buffers that inhibit RNAse action or stabilizers. [00103] In another embodiment, the kit comprises an antibody to one or more of Mindin and Hacel and a quantity of a purified standard, such as a known quantity of biomarker polypeptide.

[00104] In an embodiment, the disclosure provides a kit for detecting a biomarker comprising:

(a) one or more biomarker specific reagents that specifically bind Mindin and/or Hacel ; and

(b) instructions for use, or a quantity of purified Mindin or Hace-1 polypeptide.

[00105] In a further embodiment, the kit comprises one or more biomarker specific reagents wherein the biomarker specific reagent binds to an extracellular portion of a biomarker for example wherein the biomarker or a fragment thereof is secreted.

[00106] In an embodiment, the biomarker specific reagent is an antibody or antibody fragment.

[00107] In an embodiment, the biomarker specific reagent is a probe, or primer set that amplifies a nucleic acid transcript of the biomarker.

[00108] In an embodiment, the kit control is a peptide fragment of the at least one biomarker.

[00109] In an embodiment, the kit comprises an ELISA plate. The following non-limiting examples are illustrative of the present disclosure:

Examples

Example 1

[001 10] Mindin (Spondin 2) is a highly conserved secreted extracellular matrix (ECM) protein of the Mindin-F-spondin family. Previous studies showed Mindin to be a regulator of host innate immunity. A reduced NF-kB activation and macrophage recruitment which were accompanied by a decrease in pro-apoptotic proteins (caspase 3, 8 and 9, Bad, Bax, and Bik) and an increase in pro-survival protein of MAPK (p44/42).

[001 1 1]

Moreover, Mindin is a putative inhibitor of angiogenesis, and an increase in angiogenic factors such as vascular endothelial growth factor (VEGF) was observed post Ml. These results identify that Mindin is a significant contributor to mortality and acute adverse remodeling post infarction. This is partly through its unique attributes of innate immune regulator and inhibitor of angiogenesis. Based on these unique findings it is proposed that Mindin is a biomarker for heart diseases and/or a potent therapeutic target post Ml. Mindin exerts its positive effect by both decreasing cell death, specifically myocytes with limited proliferation capacity and also increases their survival by stimulating angiogenesis.

Example 2

[001 12] The objective of this study was to determine the role of Mindin following myocardial infarction (Ml) with loss-of-function Mindin-/- model. C57/BL wild-type (WT) mice (Figure 1) or Mindin-/- mice were randomized to permanent left anterior descending coronary artery ligation (LAD) or sham operation. Cardiac samples were collected on days 0, 3, 7, and 28 post Ml. The animals were serially followed for mortality, gene expression and physiological endpoints. Following Ml, the mortality of the Mindin-/- mice was significantly lower than that of WT mice (17.5% vs. 65.2%, p<0.0001 ). This was accompanied by a significant reduction in rupture in Mindin-/- mice (14.4% vs. 62.3%). In the WT mice, Mindin expression levels increased further on day 7 post Ml, but returned back to baseline level by day 28. Mindin-/- mice on the other hand showed significantly reduced infarct size and heart-weight/body-weight ratio compared to WT mice. Consistent with Mindin's ECM localization, cardiac matrix metalloproteinase (MMP)-9 and -2 activities in the infarcted myocardium were significantly decreased in Mindin-/- mice than in WT mice. As an innate immune regulator, Mindin-/- mice showed reduced NF-kB activation and macrophage recruitment, and these were accompanied by a decrease in pro-apoptotic proteins (caspase 3, 8 and 9, Bad, Bax, and Bik) and an increase in pro-survival protein of MAPK (p44/42) in Mindin-/- mice. Mindin is a putative inhibitor of angiogenesis, and Mindin-/- mice did show an increase in angiogenic factors such as vascular endothelial growth factor (VEGF) post Ml. We conclude that Mindin is a significant contributor to mortality and acute adverse remodeling post infarction, without wishing to be bound by theory, this may be partly through its unique attributes of innate immune regulator and inhibitor of angiogenesis. Mindin may be an interesting biomarker or therapeutic target post Ml.

Example 3

[001 13] Mindin protein levels in human serum were evaluated with Western Blot (Figure 2). Control indicates healthy individuals (n=3) and Ischemic indicates Ischemic patients (n=5). Protein expression levels were normalized against Vinculin expression. Values are mean ^*+^* SEM.

[001 14] Mindin levels in blood are increased in ischemic patients.

Example 4

[001 15] Microarray data (Table 1 and Figure 4) has shown that Mindin is increased in heart of ischemic and dilated cardiomyopathy patients. [001 16] Mindin mRNA expression in human heart tissue was evaluated with Microarray. Control indicates healthy individuals (n=3~4), ICM indicates Ischemic patients (n=3~4) and DCM indicates Dilated Cardiomyopathy Patients (n=3~4). Gene expression levels were normalized against Affimix normalization against all genes detected in Microarray. Values are mean ± SD.^* indicates the significant difference between Control and Disease group (P<0.05).

[00 7] Levels of Mindin in serum correlate with expression in heart tissue.

Example 5

[001 18] Higher expression of Mindin is detected in serum of human subjects with DCM or ICM. Mindin protein expression in human serum was evaluated with Western Blot. Control indicates healthy individuals (n=3), ICM indicates Ischemic patients (n=8) and DCM indicates Dilated Cardiomyopathy Patients (n=5). Protein expression levels were normalized against IgG Control. Values are mean ± SEM.^* indicates the significant difference between Control and Disease group (P<0.05).

Example 6

HACE1 , a novel biomarker of systolic heart failure, controls cardiac hypertrophy through regulating protein degradation

[001 19] Ppreviously a novel HECT family E3 ubiquitin-protein ligase gene, HACE1 , was identified as a potential suppressor of multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late onset tumors (Zhang, 2007). Here we report that HACEI 's gene and protein expression levels are significantly elevated in the blood samples of systolic heart failure patients (Figure 3) which indicated HACE1 could serves as a novel biomarker for distinguish systolic with diastolic heart failure. Hacel knockout mice fail to develop hypertrophy following cardiac banding. Mechanistically, HACE1 acting as a novel co-chaperone / ubiquitin ligase controls myocyte protein stability by close co-regulated with heat shock proteins. These findings also suggest that HACE1 could be a critical regulator of cardiac hypertrophy and remodelling. Example 7

[00120] A novel HECT family E3 ubiquitin-protein ligase gene, HACE1 , had previously been identified as a potential suppressor of multiple human cancers. To use as a model of human heart diseases, Hacel knock out mice were made. Hacel knockout mice fail to develop hypertrophy following pressure overload created by cardiac banding. Mechanistically, HACE1 acting as a novel co-chaperone / ubiquitin ligase controls myocyte protein stability by close co-regulated with heat shock proteins. These findings also suggest that HACE1 could be a critical regulator of cardiac hypertrophy and remodelling. Mechanistically, HACE1 acting as a novel co- chaperone / ubiquitin ligase controls myocyte protein stability by close co-regulated with heat shock proteins. These findings also suggest that HACE1 could be a critical regulator of cardiac hypertrophy and remodeling.

Example 8

Hacel Clinical utilities:

[00121] In human samples we have shown that protein expression levels are significantly elevated in the blood samples of systolic heart failure patients which indicated HACE1 could serves as a novel biomarker for distinguishing systolic with diastolic heart failure. Currently there are no biomarkers to depict the differences between these two heart failures. Moreover, based on our unique findings we propose Hacel not only as a specific biomarker to differentiate the systolic from diastolic heart diseases, but also as a potent therapeutic target post Ml.

Human blood samples:

Common name: HACE1

Map: 6q21

GenBank Accession # : AB037741

Description : HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1

Product : HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1 DBid: Gl:7243021

GO biological process : ubiquitin cycle GO molecular function : ubiquitin-protein ligase activity

GO cellular component : intracellular

RefSeq : NM_020771

UniGene : Hs.276471

Average expression level:

Rep CT 1 .183 (0.978 to 1.362)

Rep DHF 0.806 (0.637 to 1)

Rep SHF 1.248 (0.909 to 1.442)

Individual expression level:

Ind CTI 1.362

Ind CT2 0.994

lnd CT3 1.199

lnd CT4 1 .227

lnd CT5 1.137

Ind DHF1 0.96

Ind DHF2 0.785

Ind DHF3 0.676

Ind DHF4 0.656

Ind DHF5 0.78

Ind SHFI 0.921

lnd SHF2 1.324

lnd SHF3 1 .413

lnd SHF4 1.483

lnd SHF5 1.314 Table 1

Human heart tissue microarray data_gene expression level:

Rep DCM 1 .531 (1 to 2.002) [DCM: Dilated cardiomyopathy]

Rep ISC 1 .417 (0.71 1 to 1.908) [ISC: Ischemic]

Rep N 0.816 (0.616 to 0.999) [N: normal]

Rep viral 0.951 [viral]

Table 2 - Sequences

Mindin

Homo sapiens spondin 2, extracellular matrix protein (SPON2), transcript variant 2, mRNA.

ACCESSION NM_001128325

LOCUS NM_001128325 1825 bp mRNA linear 03-SEP-2009

SEQ ID NO:1 ORIGIN

1 aggagcttcc ccagccactt ccagcatg a gactgaagtc aggccacgca cagggctctc

61 caggtcacac cctgcgcaag aaggtgtagc acgctggctc taatttcgag aaggccgtgc

121 agcatcgaag acaggaggaa ctggagcctc attggccggc ccggggcgcc ggcctcgggc 181 ttaaatagga gctccgggct ctggctggga cccgaccgct gccggccgcg ctcccgctgc

241 tcctgccggg tgatggaaaa ccccagcccg gccgccgccc tgggcaaggc cctctgcgct

301 ctcctcctgg ccactctcgg cgccgccggc cagcctcttg ggggagagtc catctgttcc

361 gccagagccc cggccaaata cagcatcacc ttcacgggca agtggagcca gacggccttc

421 cccaagcagt accccctgtt ccgcccccct gcgcagtggt cttcgctgct gggggccgcg 481 catagctccg actacagcat gtggaggaag aaccagtacg tcagtaacgg gctgcgcgac

541 tttgcggagc gcggcgaggc ctgggcgctg atgaaggaga tcgaggcggc gggggaggcg

601 ctgcagagcg tgcacgcggt gttttcggcg cccgccgtcc ccagcggcac cgggcagacg

661 tcggcggagc tggaggtgca gcgcaggcac tcgctggtct cgtttgtggt gcgcatcgtg

721 cccagccccg actggttcgt gggcgtggac agcctggacc tgtgcgacgg ggaccgttgg 781 cgggaacagg cggcgctgga cctgtacccc tacgacgccg ggacggacag cggcttcacc

841 ttctcctccc ccaacttcgc caccatcccg caggacacgg tgaccgagat aacgtcctcc

901 tctcccagcc acccggccaa ctccttctac tacccgcggc tgaaggccct gcctcccatc

961 gccagggtga cactggtgcg gctgcgacag agccccaggg ccttcatccc tcccgcccca

1021 gtcctgccca gcagggacaa tgagattgta gacagcgcct cagttccaga aacgccgctg 1081 gactgcgagg tctccctgtg gtcgtcctgg ggactgtgcg gaggccactg tgggaggctc

1141 gggaccaaga gcaggactcg ctacgtccgg gtccagcccg ccaacaacgg gagcccctgc

1201 cccgagctcg aagaagaggc tgagtgcgtc cctgataact gcgtctaaga ccagagcccc

1261 gcagcccctg gggccccccg gagccatggg gtgtcggggg ctcctgtgca ggctcatgct

1321 gcaggcggcc gagggcacag ggggtttcgc gctgctcctg accgcggtga ggccgcgccg 1381 accatctctg cactgaaggg ccctctggtg gccggcacgg gcattgggaa acagcctcct

1441 cctttcccaa ccttgcttct taggggcccc cgtgtcccgt ctgctctcag cctcctcctc

1501 ctgcaggata aagtcatccc caaggctcca gctactctaa attatgtctc cttataagtt

1561 attgctgctc caggagattg tccttcatcg tccaggggcc tggctcccac gtggttgcag

1621 atacctcaga cctggtgctc taggctgtgc tgagcccact ctcccgaggg cgcatccaag 1681 cgggggccac ttgagaagtg aataaatggg gcggtttcgg aagcgtcagt gtttccatgt

1741 tatggatctc tctgcgtttg aataaagact atctctgttg ctcacaaaaa aaaaaaaaaa

1801 aaaaaaaaaa aaaaaaaaaa aaaaa

LOCUS NM 012445 1886 bp mRNA linear 03-SEP-2009

Homo sapiens spondin 2, extracellular matrix protein (SPON2),

transcript variant 1, mRNA

SEQ ID NO:2 1 gataggacag acagacaaag aaaggggtgc ggcagcactg ccaggggaag agggtgatcc

61 gacccgggga aggtcgctgg gcagggcgag ttgggaaagc ggcagccccc gccgcccccg

121 cagccccttc tcctcctttc tcccacgtcc tatctgcctc tcgctggagg ccaggccgtg

181 cagcatcgaa gacaggagga actggagcct cattggccgg cccggggcgc cggcctcggg

241 cttaaatagg agctccgggc tctggctggg acccgaccgc tgccggccgc gctcccgctg 301 ctcctgccgg gtgatggaaa accccagccc ggccgccgcc ctgggcaagg ccctctgcgc

361 tctcctcctg gccactctcg gcgccgccgg ccagcctctt gggggagagt ccatctgttc

421 cgccagagcc ccggccaaat acagcatcac cttcacgggc aagtggagcc agacggcctt

481 ccccaagcag taccccctgt tccgcccccc tgcgcagtgg tcttcgctgc tgggggccgc

541 gcatagctcc gactacagca tgtggaggaa gaaccagtac gtcagtaacg ggctgcgcga 601 ctttgcggag cgcggcgagg cctgggcgct gatgaaggag atcgaggcgg cgggggaggc

661 gctgcagagc gtgcacgcgg tgttttcggc gcccgccgtc cccagcggca ccgggcagac

721 gtcggcggag ctggaggtgc agcgcaggca ctcgctggtc tcgtttgtgg tgcgcatcgt

781 gcccagcccc gactggttcg tgggcgtgga cagcctggac ctgtgcgacg gggaccgttg

841 gcgggaacag gcggcgctgg acctgtaccc ctacgacgcc gggacggaca gcggcttcac 901 cttctcctcc cccaacttcg ccaccatccc gcaggacacg gtgaccgaga taacgtcctc

961 ctctcccagc cacccggcca actccttcta ctacccgcgg ctgaaggccc tgcctcccat 1021 cgccagggtg acactggtgc ggctgcgaca gagccccagg gccttcatcc ctcccgcccc 1081 agtcctgccc agcagggaca atgagattgt agacagcgcc tcagttccag aaacgccgct 1141 ggactgcgag gtctccctgt ggtcgtcctg gggactgtgc ggaggccact gtgggaggct 1201 cgggaccaag agcaggactc gctacgtccg ggtccagccc gccaacaacg ggagcccctg 1261 ccccgagctc gaagaagagg ctgagtgcgt ccctgataac tgcgtctaag accagagccc 1321 cgcagcccct ggggcccccc ggagccatgg ggtgtcgggg gctcctgtgc aggctcatgc 1381 tgcaggcggc cgagggcaca gggggtttcg cgctgctcct gaccgcggtg aggccgcgcc 1441 gaccatctct gcactgaagg gccctctggt ggccggcacg ggcattggga aacagcctcc 1501 tcctttccca accttgcttc ttaggggccc ccgtgtcccg tctgctctca gcctcctcct 1561 cctgcaggat aaagtcatcc ccaaggctcc agctactcta aattatgtct ccttataagt 1621 tattgctgct ccaggagatt gtccttcatc gtccaggggc ctggctccca cgtggttgca 1681 gatacctcag acctggtgct ctaggctgtg ctgagcccac tctcccgagg gcgcatccaa 1741 gcgggggcca cttgagaagt gaataaatgg ggcggtttcg gaagcgtcag tgtttccatg 1801 ttatggatct ctctgcgttt gaataaagac tatctctgtt gctcacaaaa aaaaaaaaaa 1861 aaaaaaaaaa aaaaaaaaaa aaaaaa

Human Mindin Protein SEQ ID NO:3

1 menpspaaal gkalcallla tlgaagqplg gesicsarap akysitftgk sqtafpkqy

61 plfrppaqws sllgaahssd ysmwrknqyv snglrdfaer geawalmkei eaagealqsv

121 havfsapavp sgtgqtsael evqrrhslvs f vrivpspd wfvgvdsldl cdgdrwreqa

181 aldlypydag tdsgftfssp nfatipqdtv teitssspsh pansfyyprl kalppiarvt

241 lvrlrqspra fippapvlps rdneivdsas vpetpldcev slwsswglcg ghcgrlgtks

301 rtryvrvqpa nngspcpele eeaecvpdnc

Mouse

^ fficial Symbol ^*Spon2 and ^*Name: ^*spondin 2, extracellular matrix protein [^*Mus musculus^*]

*Other Aliases: *2310045l24Rik, AI504350, M-spondin, Mindin, Mspondin ^*Chromosome: *5;* Location: ^*5 B1

*Annotation: *Chromosome 5, NC_000071.5 (33556167..33560832, complement) ^*GenelD: ^*100689

*Official Symbol ^*SPON2 and *Name: ^*spondin 2, extracellular matrix protein [^*Homo sapiens^*]

*Other Aliases: ^*DIL-1 , DILI , M-spondin, Mindin

^*Chromosome: *4;* Location: ^*4p16.3

'Annotation: ^*Chromosome 4, NC_000004.1 1 (1 160720..1 166980, complement) ^*MIM: ^*605918

*GenelD: ^*10417

The mouse mindin protein, which is 85% identical to the human protein. extracellular matrix protein [Mus muscu 330 aa SEQ ID NO:4 1 menvslalgr alwvfllami gsttsqplgg esvctarpla rysitfigkw sqtafpkqyp

61 Ifrppaqwss llgaahssdy smwrkneyvs nglrdfaerg eawalmkeie aageklqsvh

121 avfsapaips gtgqtstele vhprhslvsf vvrivpspdw fvgidsldlc eggrwkeqvv

181 ldlyphdagt dsgftfsspn fatipqdtvt eitasspshp ansfyyprlk slppiakvtf

241 vqlqqspraf appsldlasr gneivdslsv petpldcevs lwss glcgg pcgklgaksr

301 tryvrvqpan ngtpcpelee eaecapdncv spondin 2, extracellular matrix protein precursor [Homo sapiens]

NP 001121797 331 aa SEQ ID NO:5

1 menpspaaal gkalcallla tlgaagqplg gesicsarap akysitftgk wsqtafpkqy

61 plfrppaqws sllgaahssd ysmwrknqyv snglrdfaer geawalmkei eaagealqsv

121 havfsapavp sgtgqtsael evqrrhslvs fvvrivpspd wfvgvdsldl cdgdrwreqa

181 aldlypydag tdsgftfssp nfatipqdtv teitssspsh pansfyyprl kalppiarvt

241 lvrlrqspra fippapvlps rdneivdsas vpetpldcev slwss glcg ghcgrlgtks

301 rtryvrvqpa nngspcpele eeaecvpdnc v

Hacel

Human Hacel protein sequence:

LOCUS NP 065822 909 aa linear 06-AUG-2009

HECT domain and ankyrin repeat containing, E3 ubiquitin protein

ligase 1 [Homo sapiens]

ACCESSION NP 065822

HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1 [Homo sapiens] SEQ ID N0 6

ORIGIN

1 merameqlnr Itrs lrrart velpedneta vytlmpmvma dqhrsvsell snskfdvnya 61 fgrvkrsllh iaancgsvec Ivlllkkgan pnyqdisgct plhlaarngq kkcmsklley 121 sadvnicnne gltaihwlav ngrtellhdl vqhvsdvdve damgqtalhv acqnghkttv 181 qclldsgadi nrpnvsgatp lyfacshgqr dtaqilllrg akylpdkngv tpldlcvqgg 241 ygetcevliq yhprlfqtii qmtqnedlre nmlrqvlehl sqqsesqylk iltslaevat 301 tnghkllsls snydaqmks 1 Irivrmfchv frigpsspsn gidmgyngnk tprsqvfkpl 361 ellwhsldew lvliatelmk nkrdsteits illkqkgqdq daasippfep pgpgsyenls 421 tgtreskpda lagrqeasad cqdvismtan rlsaviqafy mccscqmppg mtsprfiefv 481 ckhdevlkcf vnrnpkiifd hfhfllecpe Imsrfmhiik aqpfkdrcew fyehlhsgqp 541 dsdmvhrpvn endillvhrd sifrsscevv skancaklkq giavrfhgee gmgqgwrew 601 fdilsneivn pdyalftqsa dgttfqpnsn syvnpdhlny frfagqilgl alnhrqlvni 661 yftrsfykhi lgipvnyqdv asidpeyakn lq ildndis dlgleltfsv etdvfgamee 721 vplkpgggsi lvtqnnkaey vqlvtelrmt raiqpqinaf lqgfhmfipp sliqlfdeye 781 lelllsgmpe idvsdwiknt eytsgyered pviqwfwevv editqeervl llqfvtgssr 841 vphggfanim ggsglqnfti aavpytpnll ptsstcinml klpeypskei lkdrllval 901 cgsygytma Human Hacel cDNA sequence:

LOCUS NM 020771 4576 bp mRNA linear 06-AUG-2009

Homo sapiens HECT domain and ankyrin repeat containing, E3

ubiquitin protein ligase 1 (HACE1 ) , mRNA. SEQ ID NO:7

ACCESSION NM_020771

ORIGIN

1 agaaggccac gcgagcccgg gagggacgcg gcggcggggg ctgcaggaaa ggcgcgagca

61 gaggcggcgg cgggtgtact gtaggtggtc ggtccggcag cagcccggcc cccggacgca

121 ggacgtggcc ccaggcagcc ctcgcagctc agtgctctag ccggggcaag cccgcgtctc

181 cgcctgctgg acgggcccag gcgagatgta gggctctggg cgcggaggcc gccggtgggg

241 cggctgatcg cggaggatcg cggagggcgc gccgaggatg gagagagcga tggagcaact 301 caaccgcctg acgcgctcgc tgcgccgcgc gcgcaccgtg gagttgcccg aggataatga

361 aactgctgtt tatacattaa tgccaatggt tatggctgat caacacaggt ctgtttctga

421 actactatca aattcaaaat ttgatgtcaa ttatgcattc ggacgtgtga aaagaagctt

481 gcttcacatt gcagcaaatt gtggatcggt ggaatgcttg gttttgctgt taaagaaagg

541 agcaaatcct aactatcaag atatttcagg ctgtacaccc cttcatttgg cagcaagaaa 601 tgggcagaag aaatgtatga gtaaattatt agaatatagc gctgatgtca acatttgtaa

661 taatgaaggc cttacagcaa tacattggct ggctgtgaat gggcggacag aactactcca

721 tgaccttgtg cagcatgtca gtgatgttga tgttgaggat gccatggggc agacagcact

781 gcatgttgcc tgccagaacg gtcacaagac gacagtgcag tgcttgctag acagtggtgc

841 tgatattaac aggccaaatg tatcaggagc aactccattg tactttgctt gcagtcatgg 901 tcagagagat acagcacaga tcctactatt acgaggagcc aaatatctgc cagataaaaa

961 tggagtaact cctctggatt tatgtgtaca gggtggatat ggagagactt gtgaagtatt

1021 aattcaatat cacccgaggc tttttcagac tattattcaa atgacacaga atgaagacct

1081 ccgagaaaac atgttacggc aagttctgga gcatttgtct cagcaaagtg aaagccagta

1141 cctaaagatt ctaacaagcc ttgctgaagt tgctacaaca aatggtcata aactgcttag 1201 cctctctagc aattatgatg ctcaaatgaa gagcctttta aggattgtga gaatgttttg

1261 tcacgtcttt cgaattggtc catcctcccc cagtaatgga attgatatgg gctacaatgg

1321 gaataaaact ccaagaagcc aggtgttcaa gcctctggaa ttgctttggc actcgttaga

1381 tgaatggcta gttttaatag ccacagaatt gatgaaaaac aaaagagact caacagagat

1441 cacttctatt ttactgaaac aaaaaggcca agatcaagat gctgcttcca ttcctccatt 1501 tgaacctcca ggacctggga gctatgaaaa tctgtccact ggcacaaggg aatctaaacc

1561 agatgctctt gcagggagac aggaagccag tgcagattgt caggatgtta tttctatgac

1621 agctaaccgg ctaagtgctg tcattcaagc tttttacatg tgctgttctt gtcagatgcc

1681 tccgggaatg acttcacctc gtttcattga atttgtctgc aaacatgatg aagttttaaa

1741 atgctttgtt aatagaaatc ccaaaattat atttgaccac tttcactttc tccttgaatg 1801 tcctgagttg atgtcaagat tcatgcatat cataaaagca cagcctttta aagatcgctg

1861 tgaatggttc tatgaacatt tgcattcagg acagccagat tcagatatgg tgcacaggcc

1921 agtgaatgaa aatgatatcc tgctggttca cagagattct atttttagga gtagctgtga

1981 agttgtgtca aaagcaaatt gtgcaaagct aaagcaaggg attgctgtac ggttccatgg

2041 agaagaaggc atgggtcaag gtgttgtgcg tgagtggttt gatattctgt ccaatgagat 2101 agtcaatcct gattatgcat tgtttaccca gtcagctgat ggaacaactt ttcagcctaa

2161 tagcaactct tatgtaaatc ctgatcactt gaactatttt cggtttgctg ggcagatctt

2221 gggattagcg ttgaaccaca ggcagctggt caatatttac ttcacacgat ccttctacaa

2281 gcacattctt ggtattcctg taaattacca agatgtggca tccattgatc cagaatatgc

2341 gaaaaatttg caatggattt tagataatga tataagtgat ctgggtctag aactaacttt 2401 ttctgttgag actgatgtgt ttggagcaat ggaagaggtg cctttgaaac ctgggggtgg

2461 gagtattctt gtgacacaaa ataataaagc ggagtacgtc cagcttgtta ctgaacttcg

2521 aatgacaaga gccattcagc ctcagatcaa tgctttttta cagggctttc atatgttcat

2581 tccaccctcc ctcatacagc tttttgatga atatgaattg gagctactgc tttctggcat 2641 gccagaaatt gatgtgagtg attggataaa aaatacagaa tacacaagtg gctatgaaag

2701 agaagatcca gttattcagt ggttctggga agttgtagaa gacattactc aagaggagag

2761 agttcttctc ttacagtttg ttacgggcag ttccagggtc ccacatggtg ggtttgctaa

2821 tatcatgggt ggaagtggat tgcaaaactt tacaatcgct gctgtgccat atactccaaa

2881 tcttttacca acttcaagca catgcatcaa catgctcaag ttacctgaat acccaagtaa 2941 agaaatactc aaggacagac ttcttgtggc actacattgt ggcagctatg gttacacaat

3001 ggcataatga agtctggaaa actcctctga ctactgatgc acaattcaga atggcagaag

3061 taatttggga aaatgtcaac aaaaaagcag cctaaatgca acccataggc agggctgatg

3121 cttccaattt ataaaggatc atcaggtttt ctgtttctct cttttccctt ttatgttttc

3181 tctgtttgtg atacaattag aaaatataaa atcacagtag attttatttt ttaaaatgct 3241 aactgaaagt aatagagact gtcctttttc ataattaatt ttatccaaga ttgtat aag

3301 gcaaaatctg attctacatt ccacctctgc tatgtaactg tcttgttaaa agggtgtttt

3361 ctcctaattt ctgatatatt atatgaggtc atccagctgg tgtgttcttt tgcatgtaaa

3421 ctgccattta tattttagaa aactattgta tagaatggat ttagattgtc tataaagcca

3481 caaatacgta ttttgccaca gtgtattcta tattgcaatg atttttttag cattttaata 3541 ttttaatata tattgtaaaa tttagactga tgatactaac agttgatgaa atgacatata

3601 atttatatat gaaagcttac gctatattgt atgaattatt tgcatctttc agtggccagt

3661 tttccatatg tatatattat ggtctcaatg tttttcttac gcctcatttt aatttataat

3721 gaaggtaaaa ttaaaatgta ttttaccacg tttcttttca ttacttttat ctgtgagctc

3781 tgacacatct gaaaaagtaa tctgatgtgc aaattataat ttaaatatgt taattttttt 3841 gcttcttaaa tttgcttttc atcattaaaa tgtcaagttc aagtgatatg tgcctaatat

3901 cacttggatg ttggtgggtt tttgaatttt tgggtggtta atcagtttta ttttgaaaag

3961 acgtacttga atagttacag catatgtttg aacaggaagt aggaacatgc atacacgaag

4021 aaatgctaac ggaaggattt gttatgttta ggatcttccc ttggaaacta aaaatagaat

4081 attaatgaca ttactgtttg tagaatgaca tatgcagatt ttctcataag cagtcattgt 4141 gtttgccagt aatgtttgag agacatgtaa gttgaaagtt ttgctaaatt ataaagctcc

4201 tttaattcgt tggttttgat tctcttattc tcttgtcttt tctaaatgtt aacaaaatat

4261 atcttaacag attacatgaa atttaggaat tatttaaaag ttaccattag ctctaaaatt

4321 aagattcgga tgctttattt atagtaactg aagctaataa tgttttatgt tttgattttt

4381 tgaaatttaa ttgtagaagt cactgccttc tgagttttca aatagataac cacctttaat 4441 attacactgc ttataatact aatgtttaca gatatgtttc tgtttataac catataatac

4501 attggctttg tcatattagt tttttttgca agtagttatg taaaagagat agataataaa

4561 atattaaata actgag

Mouse protein sequence for Hacel :

LOCUS NP_766061 909 aa linear 10-FEB-2008 HECT domain and ankyrin repeat containing, E3 ubiquitin protein

ligase 1 [Mus musculus] . SEQ ID NO:8

ACCESSION NP 766061

1 merameqlnr ltrslrrart velpedneta vytlmpmvma dqhrsvsell snskfdvnya

61 fgrvkrsllh iaancgsvec lvlllkkgan psyqdisgct plhlaarngq kkcmsklley

121 sadvnicnne gltaihwlav ngrtellhdl vqhvtdvdve damgqtalhv acqnghkttv

181 qclldsgadi nrpnvsgatp lyfacshgqr dtaqilllrg akylpdkngv tpldl cvqgg

241 ygqtcevliq yhprlfqtiv qmtqnedlre nmlrqvlqhl sqqsesqylk iltglaevat

301 tnghkllsls snydaqmksl lrivrifchv frigpsspsn gidmgyngnk tprsqvfkpl

361 ell hsldew lvliatelmk nkedstdits illkqkgqdq eapslsafep pgpgsyeslp

421 pgpgdskpev lageqeasad cqdvisvtan rlsaviqafy mccscqmppg mtsprfief

481 ckhdevlkcf vnrnpkiifd hfhfllecpe lmsrfmhiik aqpfkdrce fyehlhsgqp 541 dsdmvhrpvs endillvhrd sifrssceiv skancaklkq giavrfhgee gmgqgvvrew 601 fdilsneivn pdyalftqsa dgttfqpnsn syvnpdhlny frfagqilgl alnhrqlvni 661 yftrsfykhi lgipvnyqdv asidpeyakn Iqwildndis dlgleltfsv etdvfgamee 721 vplkpgggsi Ivtqnnkaey vqlvtelrmt raiqpqinaf Iqgfhmfipp sliqlfdeye 781 lelllsgmpe idvnd iknt eytsgyered pviqwfwe v editqeervl llqfvtgssr

841 vphggfanim ggsglqnfti aavpytpnll ptsstcinml klpeypskei lkdrllvalh 901 cgsygytma

Mouse cDNA sequence for Hacel :

LOCUS BC120697 3452 bp mRNA linear 07-AUG-2006

Mus musculus HECT domain and ankyrin repeat containing, E3

ubiquitin protein ligase 1, mRNA (cDNA clone MGC: 155934

IMAGE: 40129620) , complete cds .

ACCESSION BC120697 SEQ ID NO:9

ORIGIN

1 cggactgact cggcttagca tcctagcgtt cctgggcgcc ggccgcagcg agctcgctcc

61 ttctcccggc ctgtgcgggg atgtagggcg cgggtcgcgg aggccgcggg cgcggcgggg

121 gcttcgcgga ggccgcgccg aggatggaga gagcgatgga gcagctcaac cgtctgacgc 181 gctcgctgcg ccgcgcgcgc accgtggagc tgcccgagga taatgagact gctgtttaca

241 cactgatgcc aatggtgatg gccgaccagc acaggtctgt ttctgaactc ctatcaaatt

301 caaaatttga tgtcaattat gcattcggac gtgtgaagag aagcttgctt cacattgctg

361 caaactgtgg atctgtggaa tgcttggttc ttctgttgaa gaaaggcgca aaccctaact

421 atcaagatat ttcaggctgt acaccccttc atctcgctgc tagaaatgga cagaagaaat 481 gcatgagtaa attactggag tacagtgcgg atgtcaacat ttgtaataat gaaggcctta

541 cagcaataca ctggctggct gtgaatggga ggacagagct cctccatgac ctcgtgcaac

601 acgtcacaga tgtggatgtg gaggatgcca tggggcagac agcgctacac gtagcctgcc

661 agaacgggca caaaacgaca gtgcagtgct tgctagacag cggtgctgat attaacaggc

721 cgaatgtatc aggagccacg ccactgtact ttgcttgcag tcatggtcag agagatactg 781 cacagattct tctattacga ggagccaaat atttaccaga taaaaatgga gtaacccctc

841 tagatctatg tgtacagggt ggttacggac agacttgtga agtgttaatt cagtaccacc

901 cgaggctttt ccagactatt gttcaaatga cacagaatga agaccttcgg gaaaacatgt

961 tacggcaagt tctgcagcat ttgtctcagc aaagtgaaag ccagtaccta aagatcctga

1021 caggccttgc tgaagttgct acgacaaatg gccataagct gcttagttta tctagcaatt 1081 atgatgcgca gatgaagagc cttttgagga ttgtgaggat attttgtcat gtctttcgaa

1141 ttggtccctc atctcccagt aatggaatcg atatgggcta caatggaaat aaaactccaa

1201 gaagccaggt gttcaagcct ttggaattgc tttggcactc attggatgaa tggttagttt

1261 taatagccac agaattaatg aaaaacaaag aggattcaac agatattact tctatcttgc

1321 tgaaacaaaa aggccaggat caagaggccc cttctctttc tgcatttgag ccaccaggcc 1381 ctgggagcta tgaaagcctg ccccctggtc caggggactc caagccagag gtgcttgcag

1441 gggagcagga agccagtgcc gactgtcaag acgtcatctc tgtgacagcc aaccggctga

1501 gtgctgtcat tcaagccttc tacatgtgct gttcctgtca gatgcctcca ggaatgactt

1561 cacctcgttt cattgagttt gtctgcaagc atgatgaagt attgaaatgc tttgtgaata

1621 gaaaccccaa aattatattt gaccattttc actttcttct tgaatgtcct gaattgatgt 1681 caagattcat gcatatcata aaagcacagc catttaaaga tcgatgtgag tggttctatg

1741 aacatctgca ctcaggacag ccggactcag acatggtcca cagaccagtg agtgagaacg

1801 atatcctcct ggttcacaga gattctattt ttaggagtag ctgtgaaatt gtatcaaaag

1861 caaactgtgc aaagctaaag caggggattg ctgtacgatt tcatggagaa gaaggcatgg

1921 gtcaaggcgt tgtgcgtgaa tggtttgata ttctgtctaa tgagatcgtc aaccctgact 1981 atgcactgtt cacccagtca gcagacggaa caactttcca gcccaacagc aactcctatg 2041 tcaatcctga tcacttgaac tatttccggt ttgctggaca gatcttgggg ttagctttga 2101 accacaggca gctggtcaat atttacttca cacgatcatt ctacaagcac attctcggta 2161 ttcctgtaaa ttaccaagat gtagcatcta ttgatcctga atatgccaaa aatttgcaat 2221 ggattttaga taatgatatt agtgacttgg gtctagaatt gactttttct gttgagactg 2281 atgtatttgg agctatggaa gaggtgcctt taaagcctgg gggtggaagt attcttgtga 2341 cacaaaataa caaagcggag tatgtccagc tcgttactga actgcgtatg acaagagcca 2401 ttcagccgca gatcaatgcg tttttacagg gttttcatat gttcatcccg

2461 tacaactgtt tgacgagtat gaattggagc tactgctttc tggcatgccg gaaattgatg 2521 taaatgattg gataaaaaat acagaataca caagtggcta tgaaagagaa gatccagtta 2581 ttcagtggtt ctgggaagtt gtggaagaca ttactcaaga agaaagagtt cttctcttgc 2641 agttcgttac tggcagttcc agggtcccgc atggtggctt tgccaatatt atgggtggaa 2701 gtggtttgca aaactttaca attgctgctg tgccatatac tccaaatctt ttaccaactt 2761 ccagcacatg catcaacatg ctcaagttac ctgaataccc aagtaaagaa atactcaagg 2821 acagactcct tgtagcactg cattgtggca gctatggtta tacaatggca taatgaagcc 2881 tggaagactc atctgcgact gatgcacaat ccagagtgac agaagtaatt taggcatctg 2941 tcagaaaggc agcctcgcct cgacccacag gcagagagtg cctgcttaaa attcataacg 3001 aatcaggttt tctattttac atttcttttc catgttttgt ttttctatgt ttgtgataca 3061 attagaaaat ataaaatcat agtagacttc attttttaaa atgctaattg gaagtagaca 3121 cttttttttt taggtttctt taatcttatc taaaactctt aagacaaact c 1111 c c t q c 3181 attccacctc tgctgtgacg ctgtctagtt gaaagggca t ttgctgtaaa ttatatgagg 3241 acatccacct ggtgtgttct atcgcatgta aagtaccatt tatattttgg aaaaatgtaa 3301 agaatggatt aatttagatt gtgtaaagcc ataaatatgt attttcccac gttatactta 3361 tattgcaatg ggttttattt ttttgttgtt gtttagcttt ttagtatttt gatacatggt 3421 ataaagttta gactggtatg ccgaacaacg ga

While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Sequences associated with accession numbers described herein including for example the Tables, are herein specifically incorporated by reference. REFERENCES

1. Zhang, L. et al, (2007) The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers. Nat Med 13 (9), 1060-1069

Claims

Claims:

1 . A method of screening for, diagnosing or detecting heart disease in a subject, the method comprising:

a. determining a level of Mindin and/or Hacel in a sample from the subject, and

b. comparing the level of Mindin and/or Hace 1 in the sample with a control; wherein an increased level of Mindin and/or Hacel in the sample compared to the control is indicative that the subject has heart disease.

2. The method of claim 1 for treating a subject having heart disease comprising:

b. treating the subject with a treatment for heart disease when the level of Mindin in the sample is increased compared to control.

3. The method of claim 1 for monitoring response to treatment comprising:

4. The method of claim 3 for monitoring heart disease progression comprising:

b. determining a level of Mindin and/or Hace-1 in a sample taken subsequent to the base-line sample from the subject; and c. comparing the level of each biomarker in the sample with the base-line level;

wherein an increase in the Mindin and/or Hace-1 level in the post-base-line sample compared to the base-line level is indicative the heart disease is progressing, and a decrease in the biomarker level in the post base-line sample compared to the base-line level is indicative that the heart disease is not progressing.

5. The method of any one of claims 1 to 4, wherein, the level of Mindin is determined using a biomarker specific reagent, such an antibody or antibody fragment, which specifically binds Mindin.

6. A method of claim 1 for screening for, diagnosing or detecting heart disease in a subject, the method comprising:

a. determining a level of Mindin in a sample from the subject; and b. comparing the level of Mindin in the sample with a control; wherein an increased level of Mindin in the sample compared to the control is indicative that the subject has heart disease.

7. The method of claim 2 for treating a subject having heart disease comprising:

8. The method of claim 3 for monitoring response to treatment comprising:

a. determining a base-line level of Mindin in a base-line sample from the subject; b. determining a level of Mindin in a post-treatment sample from the subject; and

9. The method of claim 4 for monitoring heart disease progression comprising:

a. determining a base-line level of Mindin in a base-line sample from the subject;

c. comparing the level of each biomarker in the sample with the baseline level;

10. The method of any one of claims 1 to 9 wherein, the heart disease is ischemia, dilated cardiomyopathy or a myocardial infarction induced injury, or optionally early heart disease.

1 1. A method of claim 1 for screening for, diagnosing or detecting heart disease in a subject, the method comprising:

a. determining a level of Hacel in a sample from the subject; and b. comparing the level of Hacel in the sample with a control; wherein an increased level of Hacel in the sample compared to the control is indicative that the subject has heart disease.

12. The method of claim 2 for treating a subject having heart disease comprising: a. determining a level of Hacel in a sample from the subject, and b. treating the subject with a treatment for heart disease when the level of Hacel in the sample is increased compared to control.

13. The method of claim 3 for monitoring response to treatment in a subject being treated for heart disease comprising:

14. The method of claim 4 for monitoring heart disease progression in a subject comprising:

b. determining a level of Hacel in a sample taken subsequent to the base-line sample from the subject; and

c. comparing the level of each biomarker in the sample with the baseline level;

15. The method of any one of claims 1 to 5 or 11-14, wherein, the level of Hacel is determined using a biomarker specific reagent, such an antibody or antibody fragment, which specifically binds Hacel

16. The method of any one of claims 12 to 15 wherein the heart disease comprises systolic heart failure.

17. The method of claim 12 for distinguishing systolic heart failure from diastolic heart failure in a subject with heart failure, the method comprising:

18. The method of any one of claims 1 to 4 and 1 1 to 17 wherein the level of Hacel is determined using a biomarker specific reagent, such an antibody or antibody fragment, which specifically binds Hacel .

19. The method of any one of claims 1 to 18 wherein the biomarker level determined comprises a polypeptide level.

20. The method of any one of claims 1 to 19 wherein the sample and/or control is a blood sample.

21. The method of claim 20 wherein the blood sample comprises blood or a fraction thereof such as serum.

22. The method of any one of claims 2, 3, 7, 8 12 or 13, wherein the treatment is an agent that inhibits the biomarker such as an antibody specific for the biomarker or a siRNA molecule that inhibits the expression of the biomarker, for example injected in heart or administered intravenously.

23. A kit for detecting a biomarker for use in a method of any one of claims 1 to 22 comprising:

a. one or more biomarker specific reagents that bind Mindin or Hace ; b. a kit control; and

c. optionally instructions for use.

24. The kit of claim 23 wherein the kit control comprise a quantity of purified Mindin or Hacel polypeptide.