Patent Application
20140031291
The Sequence Listing submitted Jul. 18, 2013 as a text file named “26227—0027U2_Sequence_Listing.txt,” created on Jul. 17, 2013, and having a size of 7,069 bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).
The invention relates generally to the diagnosis, detection, and treatment of cardiovascular disease and symptoms thereof in a subject. Specifically, the invention relates to a) methods of diagnosing ischemic heart disease, non-ischemic heart disease, myocardial infarction, tachy-pacing induced non-ischemic heart disease, heart failure, cardiac arrhythmias and catecholaminergic-induced arrhythmia and symptoms thereof in a subject, b) methods of screening the efficacy of a pharmaceutical agent for treating cardiovascular disease and symptoms thereof in a subject, and c) methods of identifying a subject eligible for a cardiovascular disease treatment clinical trial.
Dysregulation of protein phosphorylation has been linked to mechanical dysfunction and arrhythmias in cardiovascular disease states including, but not limited to, atrial fibrillation, sinus node disease, heart failure, and myocardial infarction [1-5]. Emphasis has been placed on defining the levels and activities of protein kinases in cardiovascular disease. Pharmacological inhibitors of kinase activity have enhanced the treatment of cardiovascular disease phenotypes [6-8]. Beta-adrenergic receptor blockers, for example, in the treatment of arrhythmia, hypertension, and heart failure function by dampening several kinase pathways. Protein phosphatases are also responsible for regulating protein phosphorylation levels, and new therapeutic targets related to this pathway offer additional therapeutic avenues. The expression, activity, and regulation of protein phosphatases with respect to cardiovascular disease offer an opportunity to realize these new therapeutics.
Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase. The PP2A holoenzyme is comprised of three subunits: scaffolding (A subunit), regulation (B subunit), and catalytic activity (C subunit). Thus, the diversity of PP2A cellular function arises from specific combinatorial holoenzyme products (2 A subunit genes, 13 B subunit genes, and 2 C subunit genes) [9]. PP2A function is critical for the regulation of targets in both excitable and non-excitable cells including, but not limited to, ion channels and transporters, regulatory enzymes, transcription factors, and cytoskeletal proteins. Moreover, PP2A is linked to human disease. For example, PP2A dysfunction is linked with oncogenic regulation and Alzheimer's Disease [10, 11], and the design of PP2A-based inhibitors is a major area of research for cancer therapies. In myocytes, PP2A activity is linked with multiple targets important in membrane excitability and excitation-contraction coupling including ryanodine receptor (RyR2), connexin 43, Cav1.2, troponin, Na/Ca exchanger (NCX), and phospholamban [12-18]. The manipulation of PP2A activity or expression in animal or cell models produces defects in myocyte physiology and cardiac phenotypes [15, 19]. What is needed in the art are compositions and methods that determine expression of the PP2A genes, thereby detecting heart disease, and the identification targets for treatment for heart failure and arrhythmia.
Disclosed herein is a method of detecting heart disease in a subject, comprising determining in a sample from a subject expression levels of one or more Protein Phosphatase 2A (PP2A) genes and comparing the expression level of the one or more PP2A genes to expression levels of corresponding PP2A genes in a sample from a normal control, wherein an increase or decrease in the expression levels of one or more PP2A genes in the sample from the subject compared to the expression levels of corresponding PP2A genes in the sample from the normal control detects heart disease in the subject.
Also disclosed is a method of detecting heart failure in a subject, comprising determining in a sample from a subject an increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in the subject detects heart failure.
Further disclosed is a method of detecting heart failure in a subject, comprising determining in a sample from a subject a decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of methylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes in the subject detects heart failure.
Disclosed herein is a method of determining responsiveness of a heart disease in a subject to treatment with a pharmaceutical agent, comprising a) determining the expression levels of one or more PP2A genes in a first sample from the subject before treatment; b) administering to the subject the pharmaceutical agent; c) determining the expression levels of the corresponding PP2A genes in a second sample from the subject after treatment; and d) comparing the expression levels of the one or more PP2A genes in the second sample from the subject to the expression levels of the corresponding PP2A genes in the first sample from the subject, wherein the normalization of the expression levels of PP2A genes in the second sample from the subject indicates responsiveness of the heart disease to the pharmaceutical agent.
Also disclosed is a method of screening the efficacy of a pharmaceutical agent for treating heart disease in a subject, comprising a) determining the expression levels of one or more PP2A genes in a first sample from the subject before treatment; b) administering to the subject the pharmaceutical agent; c) determining the expression levels of the corresponding PP2A genes in a second sample from the subject after treatment; and d) comparing the expression levels of the one or more PP2A genes in the second sample from the subject to the expression levels of the corresponding PP2A genes in the first sample from the subject, wherein the normalization of the expression levels of PP2A genes in the second sample from the subject indicates efficacy of the pharmaceutical agent.
Further disclosed is a method of identifying a subject appropriate for a heart disease treatment clinical trial comprising determining in a sample from the subject the expression levels of one or more PP2A genes, wherein the expression levels of the one or more PP2A genes is indicative of heart disease, indicates whether the subject is appropriate for the treatment clinical trial.
Further disclosed is a method of identifying a subject appropriate for a heart disease treatment clinical trial comprising determining in a sample from a subject an increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in the subject indicates that the subject is appropriate for the treatment clinical trial.
Also disclosed is a method of identifying a subject appropriate for a heart disease treatment clinical trial comprising determining in a sample from a subject a decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of methylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes in the subject indicates that the subject is appropriate for the treatment clinical trial.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.
Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values described herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that is “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed, the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data are provided in a number of different formats and that these data represent endpoints, starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.
By “modulate” is meant to alter, by increasing or decreasing.
By “normal subject” is meant an individual who does not have heart disease.
By “normal control sample” is meant a reference sample obtained from one or more normal subjects.
As used herein, “appropriate for a treatment clinical trial” means that a subject is suitable for inclusion in the treatment clinical trial.
As used herein, the term “subject” and “patient” can be used interchangeably.
The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; E, glutamic acid.
“Peptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. For example, a peptide can be a receptor. A polypeptide is comprised of consecutive amino acids. The term “polypeptide” encompasses naturally occurring or synthetic molecules.
In addition, as used herein, the term “peptide” or “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins—Structure and Molecular Properties 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).
The phrase “nucleic acid” as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof
By an “effective amount” of a compound as provided herein is meant a sufficient amount of the compound to provide the desired effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
By “treat” is meant to administer a compound or molecule to a subject, such as a human or other mammal (for example, an animal model), that has a condition or disease, such as heart disease (for example), an increased susceptibility for developing such a disease, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease. To “treat” can also refer to non-pharmacological methods of preventing or delaying a worsening of the effects of the disease or condition, or to partially or fully reversing the effects of the disease. For example, “treat” can mean a course of action to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease other than by administering a compound.
By “prevent” is meant to minimize the chance that a subject who has susceptibility for developing disease such as heart disease will develop such a disease, or one or more symptoms associated with the disease.
By “probe,” “primer,” or “oligonucleotide” is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the “target”). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for PP2A nucleic acids (for example, genes and/or mRNAs) have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the nucleic acid to which they hybridize. Probes, primers, and oligonucleotides may be detectably-labeled either radioactively or non-radioactively, by methods well-known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction (PCR), single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, and electrophoretic mobility shift assay (EMSA). One or more of the primers or probes described herein can be used to amplify or detect one or more of the PP2A genes or gene products described herein. Additionally, the primers and probes described herein can be used in the methods described herein.
By “specifically hybridizes” is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a c-met nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.
By “high stringency conditions” is meant conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC, 0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42° C. Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well-known by those skilled in the art of molecular biology. (See, for example, F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998).
The nucleic acids, such as, the polynucleotides described herein, can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System 1Plus DNA synthesizer. Synthetic methods useful for making oligonucleotides are also described by Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994).
The full characterization of the PP2A family of polypeptides in the heart is disclosed herein. The expression of seventeen different PP2A genes in human heart and the differential expression and distribution of these genes across multiple species and different cardiac chambers were defined. The subcellular distributions of PP2A regulatory subunits in myocytes were also defined. Differential regulation of PP2A scaffolding, regulatory, and catalytic subunit expression in multiple models of cardiovascular disease as well as in human heart failure samples was found. PP2A regulation in disease extends beyond expression and subcellular location, linking differential post-translational modifications of the PP2A holoenzyme in human heart failure. Together, these findings provide new insight into the functional complexity of PP2A expression, activity, and regulation in heart and in human cardiovascular disease and identify targets for the treatment of heart failure and arrhythmia.
Disclosed herein is a gene expression panel or array for detecting heart disease, said panel or array consisting of primers or probes capable of measuring expression levels of a statistically significant number of genes of one or more of the following genes: PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, and PPP2R4. Any of the primers or probes described herein can be used in the gene expression panels or arrays for detecting heart disease as described herein. The gene expression panel or array can consist of primers or probes capable of detecting one or more genes disclosed herein. Examples of such primers and probes can be found in Table 1 of Example 1. This list is not intended to be limiting, as one of skill in the art can readily identify primers and probes for use with the compositions and methods disclosed herein, but are intended to be exemplary.
The profile of the expression levels of the genes can be used to compute a statistically significant value based on differential expression of the group of genes, wherein the computed value correlates to a diagnosis of heart disease. The variance in the obtained profile of expression levels of the said selected genes or gene expression products can be either upregulated or downregulated as compared to a control. This is described in more detail herein.
By “heart disease” is meant ischemic heart disease, non-ischemic heart disease, myocardial infarction, tachy-pacing induced and non-ischemic heart disease, cardiac arrhythmias or catecholaminergic-induced arrhythmia.
Also disclosed are diagnostic kits containing probes or primers for measuring the expression of one or more of the genes disclosed herein. For example, disclosed are diagnostic kits containing probes or primers for measuring the expression of one or more of the mRNA or protein products of: PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4. The kits can consist of primers or probes capable of detecting one or more genes disclosed herein. Examples of such primers and probes can be found in Table 1 of Example 1. This list is not intended to be limiting, as one of skill in the art can readily identify primers and probes for use with the compositions and methods disclosed herein, but are intended to be exemplary.
Disclosed herein are solid supports comprising one or more primers, probes, polypeptides, or antibodies capable of hybridizing or binding to one or more of the PP2A genes, such as PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4. Solid supports are solid-state substrates or supports with which molecules, such as analytes and analyte binding molecules, can be associated. Analytes, such as calcifying nano-particles and proteins, can be associated with solid supports directly or indirectly. For example, analytes can be directly immobilized on solid supports. Analyte capture agents, such a capture compounds, can also be immobilized on solid supports. The solid supports can consist of primers or probes capable of detecting one or more genes disclosed herein. Examples of such primers and probes can be found in Table 1 of Example 1. This list is not intended to be limiting, as one of skill in the art can readily identify primers and probes for use with the compositions and methods disclosed herein, but are intended to be exemplary.
The term “differentially expressed” or “differential expression,” as well as the term “variant,” as used herein refers to a difference in the level of expression of the biomarkers that can be assayed by measuring the level of expression of the products of the biomarkers, such as the difference in level of messenger RNA transcript or a portion thereof expressed or of proteins expressed of the biomarkers. In a preferred embodiment, the difference is statistically significant. The term “difference in the level of expression” refers to an increase or decrease in the measurable expression level of a given biomarker, for example, as measured by the amount of messenger RNA transcript and/or the amount of protein in a sample as compared with the measurable expression level of a given biomarker in a normal control. In one embodiment, the differential expression can be compared using the ratio of the level of expression of a given biomarker or biomarkers as compared with the expression level of the given biomarker or biomarkers of a normal control, wherein the ratio is not equal to 1.0. Thus, an RNA or protein is differentially expressed if the ratio of the level of expression in a first sample as compared with a second sample is greater than 1.0, for example, a ratio of greater than 1, 1.2, 1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more. Further, an RNA or protein is differentially expressed if the ratio of the level of expression in a first sample as compared with a second sample is less than 1.0., for example, less than 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. In another embodiment the differential expression is measured using p-value. For instance, when using p-value, a biomarker is identified as being differentially expressed as between a first sample and a second sample when the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, the most preferably less than 0.001.
The term “similarity in expression” as used herein means that there is no or little difference in the level of expression of the biomarkers between the test sample and the normal control or reference profile. For example, similarity can refer to a fold difference compared to a normal control. In one example, there is no statistically significant difference in the level of expression of the biomarkers.
The phrase “determining the expression of biomarkers” as used herein refers to determining or quantifying RNA or proteins or protein activities or protein-related metabolites expressed by the genes disclosed herein. The term “RNA” includes mRNA transcripts, and/or specific spliced or other alternative variants of mRNA, including anti-sense products. The term “RNA product of the biomarker” as used herein refers to RNA transcripts transcribed from the biomarkers and/or specific spliced or alternative variants. In the case of “protein,” it refers to proteins translated from the RNA transcripts transcribed from the biomarkers. The term “protein product of the biomarker” refers to proteins translated from RNA products of the biomarkers.
A person skilled in the art will appreciate that a number of methods can be used to detect or quantify the level of RNA products of the biomarkers within a sample, including arrays, such as microarrays, RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses.
Accordingly, in one example, the biomarker expression levels are determined using arrays, optionally microarrays, RT-PCR, optionally quantitative RT-PCR, nuclease protection assays or Northern blot analyses.
A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different capture compounds or detection compounds have been coupled in an array, grid, or other organized pattern.
Solid-state substrates for use in solid supports can include any solid material to which molecules can be coupled. This includes materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or a combination. Solid-state substrates and solid supports can be porous or non-porous. A form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In preferred embodiments, a multiwell glass slide can be employed that normally contains one array per well. This feature allows for greater control of assay reproducibility, increased throughput and sample handling, and ease of automation.
Different compounds can be used together as a set. The set can be used as a mixture of all or subsets of the compounds used separately in separate reactions, or immobilized in an array. Compounds used separately or as mixtures can be physically separable through, for example, association with or immobilization on a solid support. An array can include a plurality of compounds immobilized at identified or predefined locations on the array. Each predefined location on the array generally can have one type of component (that is, all the components at that location are the same). Each location will have multiple copies of the component. The spatial separation of different components in the array allows separate detection and identification of the polynucleotides or polypeptides disclosed herein.
It is not required that a given array be a single unit or structure. The set of compounds may be distributed over any number of solid supports. For example, at one extreme, each compound may be immobilized in a separate reaction tube or container, or on separate beads or microparticles. Different modes of the disclosed method can be performed with different components (for example, different compounds specific for different proteins) immobilized on a solid support.
Some solid supports can have capture compounds, such as antibodies, attached to a solid-state substrate. Such capture compounds can be specific for calcifying nano-particles or a protein on calcifying nano-particles. Captured calcifying nano-particles or proteins can then be detected by binding of a second detection compound, such as an antibody. The detection compound can be specific for the same or a different protein on the calcifying nano-particle.
Methods for immobilizing nucleic acids, peptides or antibodies (and other proteins) to solid-state substrates are well established Immobilization can be accomplished by attachment, for example, to aminated surfaces, carboxylated surfaces or hydroxylated surfaces using standard immobilization chemistries. Examples of attachment agents are cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents, epoxides and maleimides. A preferred attachment agent is the heterobifunctional cross-linker N-[γ-Maleimidobutyryloxy]succinimide ester (GMBS). These and other attachment agents, as well as methods for their use in attachment, are described in Protein immobilization: fundamentals and applications, Richard F. Taylor, ed. (M. Dekker, New York, 1991);, Johnstone and Thorpe, Immunochemistry In Practice (Blackwell Scientific Publications, Oxford, England, 1987) pages 209-216 and 241-242, and Immobilized Affinity Ligands; Craig T. Hermanson et al., eds. (Academic Press, New York, 1992) which are incorporated by reference in their entirety for methods of attaching antibodies to a solid-state substrate. Antibodies can be attached to a substrate by chemically cross-linking a free amino group on the antibody to reactive side groups present within the solid-state substrate. For example, antibodies may be chemically cross-linked to a substrate that contains free amino, carboxyl, or sulfur groups using glutaraldehyde, carbodiimides, or GMBS, respectively, as cross-linker agents. In this method, aqueous solutions containing free antibodies are incubated with the solid-state substrate in the presence of glutaraldehyde or carbodiimide.
A method for attaching antibodies or other proteins to a solid-state substrate is to functionalize the substrate with an amino- or thiol-silane, and then to activate the functionalized substrate with a homobifunctional cross-linker agent such as (Bis-sulfo-succinimidyl suberate (BS3) or a heterobifunctional cross-linker agent such as GMBS. For cross-linking with GMBS, glass substrates are chemically functionalized by immersing in a solution of mercaptopropyltrimethoxysilane (1% vol/vol in 95% ethanol pH 5.5) for 1 hour, rinsing in 95% ethanol and heating at 120° C. for 4 hrs. Thiol-derivatized slides are activated by immersing in a 0.5 mg/ml solution of GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at room temperature. Antibodies or proteins are added directly to the activated substrate, which are then blocked with solutions containing agents such as 2% bovine serum albumin, and air-dried. Other standard immobilization chemistries are known by those of skill in the art.
Each of the components (compounds, for example) immobilized on the solid support preferably is located in a different predefined region of the solid support. Each of the different predefined regions can be physically separated from each other of the different regions. The distance between the different predefined regions of the solid support can be either fixed or variable. For example, in an array each of the components can be arranged at fixed distances from each other, while components associated with beads will not be in a fixed spatial relationship. In particular, the use of multiple solid support units (for example, multiple beads) will result in variable distances.
Components can be associated or immobilized on a solid support at any density. Components preferably are immobilized to the solid support at a density exceeding 400 different components per cubic centimeter. Arrays of components can have any number of components. For example, an array can have at least 1,000 different components immobilized on the solid support, at least 10,000 different components immobilized on the solid support, at least 100,000 different components immobilized on the solid support, or at least 1,000,000 different components immobilized on the solid support.
Optionally, at least one address on the solid support can be a probe specific for one or more of the genes disclosed herein. Disclosed are solid supports where at least one address is the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein. Solid supports can also contain at least one address that is a variant of the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Solid supports can also contain at least one address that is a variant of the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein.
In addition, the genes described herein may be used as markers for presence or progression of heart disease. The methods and assays described elsewhere herein may be performed over time, and the change in the level of reactive polypeptide(s), polynucleotide(s), and or polypeptide “activity” evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. Assays can be performed prior to, during, or after a treatment protocol. As noted herein, to improve sensitivity, multiple genes may be assayed within a given sample. Binding agents specific for different proteins, antibodies, nucleic acids thereto provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of receptors may be based on routine experiments to determine combinations that results in optimal sensitivity. To assist with such assays, specific biomarkers can assist in the specificity of such tests. As such, disclosed herein is a biomarker, wherein the biomarker is capable of binding to or hybridizing with one or more genes or peptides as disclosed herein.
According to a further aspect, there is provided a computer implemented product for detecting heart disease, comprising (a) a means for receiving values corresponding to a subject expression profile in a subject sample; and (b) a database comprising a reference expression profile associated with a diagnosis or prognosis, wherein the subject biomarker expression profile and the biomarker reference profile each have at least three values representing the expression level of at least one biomarker selected from PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4, wherein the computer implemented product selects the biomarker reference expression profile most similar to the subject biomarker expression profile, to thereby diagnose or prognose the subject.
Preferably, a computer implemented product described herein is for use with a method described herein.
Preferably, the data structure is capable of configuring a computer to respond to queries based on records belonging to the data structure, each of the records comprising: (a) a value that identifies a biomarker reference expression profile of at least one gene selected from PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 and (b) a value that identifies the probability of a diagnosis associated with the biomarker reference expression profile.
According to a further aspect, there is provided a computer system comprising (a) a database including records comprising a biomarker reference expression profile of at least one gene selected from PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4, associated with a diagnosis of heart disease; (b) a user interface capable of receiving a selection of gene expression levels of the at least one gene for use in comparing to the biomarker reference expression profile in the database; and (c) an output that displays a diagnosis or prognosis or therapy according to the biomarker reference expression profile most similar to the expression levels of the at least one gene.
In a further aspect, the application provides computer programs and computer implemented products for carrying out the methods described herein. Accordingly, in one embodiment, the application provides a computer program product for use in conjunction with a computer having a processor and a memory connected to the processor, the computer program product comprising a computer readable storage medium having a computer mechanism encoded thereon, wherein the computer program mechanism may be loaded into the memory of the computer and cause the computer to carry out the methods described herein.
The disclosed genes and peptides, including PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 can be used in a variety of different methods, for example in prognostic, predictive, diagnostic, and therapeutic methods and as a variety of different compositions.
Disclosed herein is a method of detecting heart disease in a subject, comprising determining in a sample from a subject expression levels of one or more PP2A genes and comparing the expression level of the one or more PP2A genes to expression levels of corresponding PP2A genes in a sample from a normal control, wherein an increase or decrease in the expression levels of one or more PP2A genes in the sample from the subject compared to the expression levels of corresponding PP2A genes in the sample from the normal control detects heart disease in the subject.
The expression level of the one or more PP2A genes is determined by amplifying a nucleic acid sample obtained from the subject. The method can also further comprise administering a therapeutic composition to the subject.
The PP2A genes can be catalytic subunit genes, such as PPP2CA or PPP2CB. One can measure an increase in the expression level of PPP2CA and PPP2CB in the subject compared to the expression levels of PPP2CA and PPP2CB in the normal control to detect heart disease in the subject. One can then further administer a therapeutic composition to the subject.
The PP2A genes can also be scaffolding subunit genes, such as PPP2R1A or PPP2R1B. Disclosed herein is a method of determining an increase in the expression level of PPP2R1A and PPP2R1B in the subject compared to the expression levels of PPP2R1A and PPP2R1B in the normal control thereby detecting heart disease in the subject. One can then further administer a therapeutic composition to the subject.
The PPP2A genes can be regulatory subunit genes, such as PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4. Disclosed herein is a method of determining increased expression levels of one or more of PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, and PPP2R4 in the subject compared to the expression levels of the corresponding genes in the normal control detect heart disease. One can then further administer a therapeutic composition to the subject. One can determine the expression levels of one or more of PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, and PPP2R4 in the subject compared to the expression levels of the corresponding genes in the normal control to detect heart disease. One can then administer a therapeutic composition to the subject.
Also disclosed herein is a method wherein increased expression levels of PPP2CA, PPP2CB, PPP2R5A, PPP2R5B, PPP2R5E, PPP2R3A, and PPP2R4 compared to the expression levels of the corresponding genes in the normal control detects ischemic heart disease in the subject. One can then administer a therapeutic composition to the subject.
Also disclosed is a method of determining an increased level of expression of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R5A, PPP2R5B, PPP2R5E, PPP2R3A, and PPP2R4 compared to the expression levels of the corresponding genes in the normal control to detect non-ischemic heart disease in the subject. One can then administer a therapeutic composition to the subject.
Also disclosed is a method of determining increased levels of expression of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R5D, and PPP2R4, and a decreased level of expression of PPP2R5A compared to the expression levels of the corresponding genes in the normal control to detect myocardial infarction in the subject. One can then administer a therapeutic composition to the subject.
The heart disease can be tachy-pacing induced non-ischemic heart disease, for example. Thus, disclosed is a method of determining increased levels of expression of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2B, PPP2R5B, PPP2R5D, PPP2R5E, and PPP2R4 compared to the expression levels of the corresponding genes in the normal control to detect tachy-pacing induced non-ischemic heart disease in the subject. One can then administer a therapeutic composition to the subject.
The heart disease can be catecholaminergic induced arrhythmia. Thus, disclosed is a method of determining increased levels of expression of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R5A, and PPP2R5B, and a decreased level of expression of PPP2R5E compared to the expression levels of the corresponding genes in the normal control to detect cardiac arrhythmias or catecholaminergic induced arrhythmia in the subject. One can then administer a therapeutic composition to the subject.
“Biological sample” as used herein means a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue or fluid isolated from subjects. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, blood fraction, plasma, serum, sputum, stool, tears, transudates, exudates, breast milk, prostatic fluid, semen, mucus, hair, skin, urine, effusions, ascitic fluid, amniotic fluid, saliva, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions, cell line, tissue sample, or secretions. A biological sample may be provided by removing a sample of cells from a subject but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Biological samples also include explants and primary and/or transformed cell cultures derived from animal or human tissues.
As used herein, the term “subject” refers to a mammal, including both human and other mammals. The methods disclosed herein can be applied to human subjects.
Also disclosed is a method of detecting heart failure in a subject, comprising determining in a sample from a subject an increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in the subject detects heart failure.
The ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes can be determined by performing measurements of each form using selective antibodies versus ratios of total protein levels and housekeeping “control” polypeptides by immunoblot.
Also disclosed is a method of detecting heart failure in a subject, comprising determining in a sample from a subject a decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of methylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes in the subject detects heart failure. The ratio of methylated catalytic subunit genes to total catalytic subunit genes can be determined by performing measurements of each form using selective antibodies versus ratios of total protein levels and housekeeping “control” polypeptides by immunoblot.
As the term is used herein, an “increased ratio” refers to an identified, reproducible, quantitative or qualitative, increase in the relative amount of the physical presence of one or more of the following: PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4, as compared with another target or with housekeeping genes including, but not limited to cardiac gene products including actin and GAPDH that display minimal to no change in heart disease phenotypes. Housekeeping gene products may differ based on the specific cardiac pathology.
As the term is used herein, a “decreased ratio” refers to an identified, reproducible, quantitative or qualitative, decrease in the relative amount of the physical presence of one or more of the following: PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4, as compared with another target or with housekeeping genes including, but not limited to cardiac gene products including actin and GAPDH that display minimal to no change in heart disease phenotypes. Housekeeping gene products may differ based on the specific cardiac pathology.
The relative amount of the respective entities is determined by quantitative measurement of the two entities (e.g., a specifically phosphorylated OR methylated molecules in a target cell or tissue) by appropriately comparable methods of detection, with calculation of the relative ratio by standard mathematical means (e.g., determination of the ratio of the two determined measurements) to produce a quantitative measurement reflective of the relative amount of the two entities. The identical methods are used to measure the relative ratio of the same respective entities in an appropriate control cell or tissue, to produce a quantitative measurement reflective of the relative amount of the two entities under normal, non-disease conditions. An increase in the relative ratio is identified by comparison of the relative amount calculated from the target tissue to the relative amount calculated from the normal control tissue, wherein a reproducible, statistically significant, higher relative amount is quantitatively or qualitatively determined. A decrease in the relative ratio is identified by comparison of the relative amount calculated from the target tissue to the relative amount calculated from the normal control tissue, wherein a reproducible, statistically significant, lower relative amount is quantitatively or qualitatively determined.
It will be appreciated that in the methods described herein that involve comparison of a determined level or amount to a reference or normal control amount, standard methods available to the skilled practitioner can be used to obtain the appropriate reference amounts. In one embodiment, the reference amount is obtained from a reference or control sample obtained from the subject (e.g. a biological sample). Typically, normal, healthy tissue, (e.g., located adjacent to the sample tissue), is used as the reference profile or normal control tissue. Preferably the control tissue, or biological sample, is of the same type as the tested sample tissue. In certain embodiments, wherein the progression of heart disease in a subject is to be monitored over time, the reference or control sample can be the tested sample tissue taken from the subject at an earlier date.
In some embodiments, the reference can be a level of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 in a normal healthy subject with no symptoms or signs of heart disease. Preferably the reference is of the same tissue type as the tested sample. For example, a normal healthy subject has normal heart tissue morphology, and/or is not diagnosed with heart disease, and/or has been identified as having a genetic predisposition for developing heart disease (e.g. a family history of heart disease or a genetic test for genotypes associated with an increased risk of heart disease) and/or has been exposed to risk factors for heart disease (e.g. smoke, cigarette smoke, alcohol, tobacco, obesity, etc). In some embodiments, the reference can also be a level of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
Methods to measure, detect or determine PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 are well known to a skilled artisan. The genes or the encoded proteins can be measured or detected. For example, an increase in PPP2CA can be determined by measuring the PPP2CA gene expression levels or the PPP2CA protein levels. Any of the disclosed methods of detecting heart disease in a subject, detecting heart failure in a subject, determining responsiveness of a subject to a treatment, screening for efficacy of pharmaceutical agents, and identifying subjects for clinical trial can use the methods or assays of measuring or detecting the PP2A genes or proteins described herein.
Methods of measuring or detecting gene expression are well known in the art. Assays such as but not limited to reverse transcription-PCR, real time PCR, and northern blots can be used. Reverse transcription-PCR allows for the amplification of cDNA produced from RNA from a particular sample. The RNA reflects the gene expression and is ultimately amplified after being converted to cDNA. For example, gene expression can be measured by obtaining a sample containing mRNA from a subject. Using reverse transcriptase, the mRNA can be converted to cDNA, thus making a complimentary DNA strand. The cDNA can then be amplified using common amplification techniques. Real-time PCR allows for the direct quantification of DNA in real time (i.e. during amplification). Northern blots provide the ability to determine the presence and quantification of RNA in a sample by hybridizing the RNA with a specific probe that binds to the nucleic acid sequence of interest. The primers and probes used to detect specific nucleic acids (RNA, DNA or cDNA) can be determined by one of skill in the art based on the sequence of the gene to be detected. For example, the primers used for reverse transcription-PCR can be short nucleic acid sequences that are complementary to sequences of a particular gene, such as PPP2CA. Any of the primers identified in Table 1 can be used to detect or measure gene expression of the PP2A genes.
In one embodiment, methods of measurement involve use of a binding protein (e.g. an antibody) for specific detection (e.g., immunodetection) of the specified protein. Such methods to measure protein level include ELISA (enzyme linked immunosorbent assay), Western blot, proteomic microarray, immunoprecipitation, immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject is detected by standard imaging techniques. Such antibody-based methods can distinguish between the proteins disclosed herein. Antibodies specific for the various isoforms of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 are available in the art.
The techniques of immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) are particularly suitable for use in the methods described herein. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction and thereby experience a change (e.g., color or light emission), upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, which includes the marker stain or marker signal, follows the application of a primary specific antibody. In some embodiments, the level of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 can be determined using a staining intensity scale and immunohistochemistry as described in the Examples herein.
The technique of proteomic microarray analysis can be used in the methods described herein. Proteomic microarrays are based on miniature arrays of ligands. Proteins in a sample bind to the ligands on the array and are detected and the amount quantified, often by using fluorescent tags (Templin et al. Proteomics 2003 3:2155-2166; MacBeath, Nature Genetics 2002 32:526-532).
In addition, protein levels may be detected using Mass Spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference. Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such as proteins (see, e.g., Li et al. (2000) Tibtech 18:151-160; Rowley et al. (2000) Methods 20: 383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8: 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al., Science 262:89-92 (1993); Keough et al., Proc. Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000). In certain embodiments, a gas phase ion spectrophotometer is used.
In other embodiments, laser-desorption/ionization mass spectrometry is used to analyze the level of a protein. Modern laser desorption/ionization mass spectrometry (“LDI-MS”) can be practiced in two main variations: matrix assisted laser desorption/ionization (“MALDI”) mass spectrometry and surface-enhanced laser desorption/ionization (“SELDI”). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological molecules without significantly fragmenting them. However, MALDI has limitations as an analytical tool. It does not provide means for fractionating the sample, and the matrix material can interfere with detection, especially for low molecular weight analytes. See, e.g., U.S. Pat. No. 5,118,937 (Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait). In SELDI, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest. In another variant, the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser. In another variant, the surface is derivatized with molecules that bind the protein of interest and that contain a photolytic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied. See, e.g., U.S. Pat. No. 5,719,060 and WO 98/59361. The two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix-containing liquid to the captured analyte to provide the energy absorbing material.
For additional information regarding mass spectrometers, see, e.g., Principles of Instrumental Analysis, 3rd edition., Skoog, Saunders College Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094. Detection of the presence of AID mRNA or protein will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of a polypeptide bound to the substrate. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g., visually, by computer analysis, etc.), to determine the relative amounts of particular biomolecules. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.
Also disclosed is a method of determining responsiveness of a heart disease in a subject to treatment with a pharmaceutical agent, comprising a) determining the expression levels of one or more PP2A genes in a first sample from the subject before treatment; b) administering to the subject the pharmaceutical agent; c) determining the expression levels of the corresponding PP2A genes in a second sample from the subject after treatment; and d) comparing the expression levels of the one or more PP2A genes in the second sample from the subject to the expression levels of the corresponding PP2A genes in the first sample from the subject, wherein the normalization of the expression levels of PP2A genes in the second sample from the subject indicates responsiveness of the heart disease to the pharmaceutical agent.
Also disclosed is a method of screening the efficacy of a pharmaceutical agent for treating heart disease in a subject, comprising a) determining the expression levels of one or more PP2A genes in a first sample from the subject before treatment; b) administering to the subject the pharmaceutical agent; c) determining the expression levels of the corresponding PP2A genes in a second sample from the subject after treatment; and d) comparing the expression levels of the one or more PP2A genes in the second sample from the subject to the expression levels of the corresponding PP2A genes in the first sample from the subject, wherein the normalization of the expression levels of PP2A genes in the second sample from the subject indicates efficacy of the pharmaceutical agent.
As described herein, disclosed are methods of detecting heart disease in a sample comprising determining the expression level of one or more genes in a sample and comparing those expression levels to the expression levels of a normal control sample, wherein the expression level of one or more genes or peptides is increased or decreased by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression level of a “normal” control sample is indicative of a heart disease. In addition, the expression level of one or more genes or peptides can be increased or decreased by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the expression level of a “normal” control sample is indicative of heart disease.
Another aspect of the invention relates to a method for diagnosing whether a subject has an increased likelihood of having heart disease, the method comprising determining the level of expression of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 in the subject, in heart tissue of the subject or in a biological sample obtained from the subject, and comparing to the level of expression in an appropriate reference sample or normal control sample, wherein a reduction or increase of the level as specified is indicative of the subject having an increased risk of having heart disease at the time the method is performed.
When the subject is identified to be at risk of developing heart disease using the methods as disclosed herein, the subject may develop the heart disease in the near future or anytime in the future. Accordingly, such subjects can be selected for frequent follow up measurements of the levels of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4, to allow early treatment of the heart disease. Alternatively, the present invention provides methods to diagnose subjects who are at a lesser risk of developing heart disease by analyzing the levels of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 to identify subjects at no or minimal risk of heart disease, who can be selected to undergo less frequent follow up measurements of the level, or other alternative invasive or non-invasive diagnostic methods.
An increase or decrease in the expression level of the genes or peptides disclosed herein is not always required to indicate heart disease. There can be signature patterns of increased or decreased expression levels of one or more of the genes or peptides.
The variance in the obtained profile of expression levels of the said selected genes or gene expression products in said subject's sample can be used to determine the type of treatment, or combination of treatments, that the subject should receive. Examples of treatments typically given to subjects diagnosed with heart disease include, but are not limited to, aspirin, diuretics, calcium channel inhibitors, antiarrhythmics, catecholamines and catecholamine receptor agonists and antagonists (including α- and β-adrenergic receptor agonists and antagonists), vasopressin receptor agonists and antagonists, organic nitrates, activators of soluble and particulate guanylyl cyclases, natriuretic peptides or other components that bind to natriuretic peptide receptors and stimulate or inhibit guanylyl cyclase activity, renin inhibitors, angiotensin-converting enzymes inhibitors, and angiotensin receptor antagonists.
Also disclosed is a method of identifying a subject appropriate for a heart disease treatment clinical trial comprising determining in a sample from the subject the expression levels of one or more PP2A genes, wherein the expression levels of the one or more PP2A genes is indicative of heart disease, indicates whether the subject is appropriate for the treatment clinical trial. The expression levels of one or more PP2A genes can be determined by amplifying a nucleic acid sample obtained from the subject. An increase in the expression levels of PPP2CA or PPP2CB in the subject compared to the expression levels of PPP2CA or PPP2CB in a normal control can indicate the subject is appropriate for the treatment clinical trial.
Also disclosed is a method wherein an increase in the expression levels of PPP2R1A or PPP2R1B in the subject compared to the expression levels of PPP2R1A or PPP2R1B in the normal control, indicates the subject is appropriate for the treatment clinical trial.
An increased expression level of one or more of PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, and PPP2R4 in the subject compared to the expression levels of the corresponding genes in the normal control can indicate the subject is appropriate for the treatment clinical trial. A decreased expression level of one or more of PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, and PPP2R4 in the subject compared to the expression levels of the corresponding genes in the normal control can indicate the subject is appropriate for the treatment clinical trial.
Also disclosed is a method of identifying a subject appropriate for a heart disease treatment clinical trial comprising determining in a sample from a subject an increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the increased ratio of phosphorylated catalytic subunit genes to total catalytic subunit genes in the subject indicates that the subject is appropriate for the treatment clinical trial.
Also disclosed is a method of identifying a subject appropriate for a heart disease treatment clinical trial comprising determining in a sample from a subject a decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes compared to a ratio of methylated catalytic subunit genes to total catalytic subunit genes in a normal control sample, wherein the decreased ratio of methylated catalytic subunit genes to total catalytic subunit genes in the subject indicates that the subject is appropriate for the treatment clinical trial.
In one aspect, the methods can be used to ensure patients are identified to participate in treatment clinical trials based upon the likelihood of heart disease and whether subject can benefit from treatment to prevent the development of heart disease. In one aspect, the methods can comprise measuring an initial value of PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R2C, PPP2R2D, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, PPP2R5E, PPP2R3A, PPP2R3B, PPP2R3C, or PPP2R4 in a subject before the subject has received treatment. Repeat measurements can then be made over a period of time. For example, and not to be limiting, that period of time can be about 1 day, 2 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, or greater than 1 year. If the initial level is elevated relative to the average level in a control population, a significant reduction in level in subsequent measurements can indicate a positive treatment outcome. Likewise, if the initial level of a measure marker is reduced relative to the average in a control population, a significant increase in measured levels relative to the initial level can signal a positive treatment outcome. Subsequently measured levels are considered to have changed significantly relative to initial levels if a subsequent measured level differs by more than one standard deviation from the mean of repeat measurements of the initial level. If monitoring reveals a positive treatment outcome, that indicates a patient that can be chosen to participate in a treatment clinical trial for that particular therapeutic agent or agents. If monitoring reveals a negative treatment outcome, that indicates a patient that should not be chosen to participate in a treatment clinical trial for that particular therapeutic agent or agents.
Methods of determining or identifying subjects eligible to participate in clinical trial treatments can include the steps of enrolling the subject in the clinical trial and administering the treatment to the subject. For example, after identifying a subject appropriate for a heart disease treatment clinical trial, the subject can be administered a heart disease treatment such as but not limited to aspirin, diuretics, calcium channel inhibitors, antiarrhythmics, catecholamines and catecholamine receptor agonists and antagonists (including α- and β-adrenergic receptor agonists and antagonists), vasopressin receptor agonists and antagonists, organic nitrates, activators of soluble and particulate guanylyl cyclases, natriuretic peptides or other components that bind to natriuretic peptide receptors and stimulate or inhibit guanylyl cyclase activity, renin inhibitors, angiotensin-converting enzymes inhibitors, and angiotensin receptor antagonists.
Also disclosed are methods of treating a subject having heart disease comprising diagnosing or detecting heart disease in a subject using any of the methods disclosed herein, and administering a pharmaceutical agent for treating heart disease to a subject having heart disease or ceasing any treatment for heart disease in a subject not having heart disease. The pharmaceutical agent can be any agent known to treat heart disease or can be a pharmaceutical agent that alters PP2A gene expression. For example, pharmaceutical agents determined with the disclosed screening assays can be used in the methods of treating a subject having heart disease.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
The human genome encodes over seventeen different PP2A subunits (
Human tissue samples. Left ventricular (LV) tissue was obtained from explanted hearts of patients undergoing heart transplantation through The Cooperative Human Tissue Network: Midwestern Division at OSU. Approval for the use of human subjects was obtained from the Institutional Review Board of OSU. LV tissue from healthy donor hearts not suitable for transplantation was obtained through the Iowa Donors Network and the National Disease Research Interchange. The investigation conformed to the principles outlined in the Declaration of Helsinki. Age and sex were the only identifying information acquired from tissue providers.
mRNA analysis. PCR reactions were performed in a volume of 20 μl using Platinum Taq Polymerase High Fidelity. A touchdown PCR protocol was used for each primer set to reduce any non-specific sequence amplification. See Table 1 for primer sequences.
PP2A subunit gene product expression in human heart was also tested. Specifically, the protein expression of catalytic, regulatory, and scaffolding subunits was tested by mRNA (
Finally, whether subtype expression was conserved across species was tested. Immunoblots of human, canine, rat, and mouse LV confirmed the presence of both the PP2A catalytic and scaffolding subunits (PP2A-A, PP2A-C) as well as regulatory subunits PPP2R2A, PPP2R2B, PPP2R3A, PPP2R4, PPP2R5A, PPP2R5B, PPP2R5C, PPP2R5D, and PPP2R5E (
Immunoblots. Following quantification, tissue lysates were analyzed on Mini-PROTEAN tetra cell (BioRad) on a 4-15% precast TGX gel (BioRad). Gels were transferred to a nitrocellulose membrane using the Mini-PROTEAN tetra cell (BioRad). Membranes were blocked for 1 hour at room temperature using a 3% BSA solution and incubated with primary antibody overnight at 4° C. Antibodies included: anti-PP2A-C subunit (1:500, Millipore 05-421), anti-PPP2R5E (1:1000, Sigma HPA006034), anti-PP2A subunit B isoform PR55α (1:500, Sigma SAB4200241), anti-PP2A subunit B isoform B56-δ (Sigma SAB4200255), anti-PPP2R5A (1:500, Abcam ab72028) or anti-PP2A-B56-α (1:200, Santa Cruz sc-136045), anti-PPP2R5C (1:500 Abcam ab94633), anti-PP2A alpha phospho Y307 (1:500 Abcam, ab32104), anti-PP2A methyl L309 (1:500, Abcam ab66597), anti-PPP2R5B (Abcam ab1366), anti-PPP2R3A (Sigma HPA035829), and anti-PP2A/A (Calbiochem 539509). Secondary antibodies included donkey-anti-mouse-HRP, donkey-anti-rabbit-HRP, and donkey-anti-goat-HRP (Jackson Laboratories). Densitometry was performed using Adobe Photoshop software and all data were normalized to GAPDH levels present in each sample.
Tissue preparation. Cardiac tissue from human, canine, and murine hearts was flash frozen with liquid N2 and ground into a fine powder using a chilled mortar and pestle. The resulting powder was then resuspended in homogenization buffer (1 mM NaHCO3, 5 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 1 mMNaF, 1 mM PMSF, and protease inhibitor cocktail [Sigma]) and further homogenized using a chilled Dounce homogenizer. Samples were flash frozen in liquid N2 and stored at −80° C. for immunoblots.
Whether a secondary level of target specificity arises through the subcellular distribution of specific PP2A holoenzymes was tested by evaluating the subcellular localization of PP2A subunits in primary adult cardiomyocytes. Broad cellular distribution of PP2A scaffolding and catalytic subunits throughout the cytosol and nucleus of ventricular cardiomyocytes was observed. In fact, immunostaining for both the A and C subunits was present across the entire myocyte (
Immunofluorescence. Isolated cardiomyocytes were blocked in PBS, containing 0.075% Triton X-100 and 3% BSA, and were incubated in primary antibody overnight at 4° C. Following washes (PBS plus 0.1% Triton X-100), the slides were incubated in secondary antibody (Alexa 488, or 568) for 4 hours at 4° C. and mounted using Vectashield (Vector) and coverslips. Images were collected on a Zeiss 510 Meta confocal microscope (40 power oil 1.40 NA (Zeiss), pinhole equal to 1.0 Airy Disc) using Carl Zeiss Imaging software. Images were imported into Adobe Photoshop for cropping and linear contrast adjustment.
Cardiomyocyte preparations. Neonatal and adult cardiomyocytes were prepared as previously described [27, 20].
Whether the expression levels of PP2A subunits were altered in multiple human heart failure pathologies was determined. In particular, whether variability in the expression of the regulatory subunits serves as a molecular identifier for disease was determined PP2A subunit expression in human ischemic heart failure samples was first determined. While PP2A scaffolding subunit levels were unaffected in LV tissue, nearly a two fold increase in PP2A catalytic subunit expression, when compared to control hearts, was observed (n=5 control and non-ischemic, p<0.05). Differential regulation of regulatory subunits in ischemic heart failure was also observed, with elevated levels of PPP2R5A, PPP2R5B, PPP2R5E, PPP2R3A, and PPP2R4 (
PP2A subunit expression was examined in LV samples from both non-ischemic and control hearts. While similar data for PP2A catalytic subunit (increased approximately two fold) were observed, PP2A scaffolding subunit levels were also elevated in non-ischemic LV (n=5 control and non-ischemic, p<0.05 for A, C subunits). Regulatory subunit overexpression was observed for PPP2R5A, PPP2R5B, PPP2R5E, PPP2R3A, and PPP2R4, similar to ischemic heart failure data (
PP2A subunit expression in two different canine models of human heart disease was examined PP2A subunit expression following myocardial infarction (through coronary artery occlusion), a leading cause of death worldwide, was first evaluated [23]. Similar to both ischemic and non-ischemic human heart failure samples, elevated levels of PP2A catalytic subunit were observed in infarct border zone tissue compared to remote tissue of a well validated canine myocardial infarction model [24] (
In addition to the canine MI model above, PP2A subunit expression was also evaluated in a well validated long-term tachy-pacing induced non-ischemic model of canine heart failure [22]. At four months of chronic pacing, elevated levels of both the scaffolding and catalytic subunits were observed in experimental samples compared with control tissue (
Canine ischemic and non-ischemic heart failure models. Myocardial infarction was produced in healthy mongrel dogs by total coronary artery occlusion, as described previously [20]. A cardiectomy was performed five days after surgery. Thin tissue slices from visible epicardial border zone (BZ) and from a remote area away from the infarct (LVbase) were flash frozen for analysis. HF via chronic tachypacing was induced as described [21, 22]. Cardiac tissues were snap frozen in liquid nitrogen and stored at −80° C. until used. Control dogs were sacrificed in parallel.
Over the past decade, dysregulation in cardiac sympathetic tone has been linked to both genetic and acquired forms of human ventricular arrhythmia [14]. In fact, mutations in select myocyte pathways that alter the phospho-protein axis have been identified as a primary cause of multiple forms of congenital catecholaminergic polymorphic ventricular tachycardia (CPVT) [25]. One notable pathway altered in human CPVT, sinus node disease, heart rate variability, and atrial fibrillation is the ankyrin-B-pathway that coordinates the subcellular localization of membrane-associated ion channels, transporters, and signaling molecules [26-29]. Whether PP2A subunit levels were altered in an animal model for human ankyrin-B CPVT was examined Consistent with investigated forms of human and canine heart disease (
Mouse heart failure model with proximal aortic banding. Adult male B6 mice were subjected to pressure overload by TAC surgery as previously described [21].
While tissue, cell, and subcellular subunit expression and localization play key roles in determining local holoenzyme function, post-translational regulation of PP2A activity has been implicated in holoenzyme regulation in other organ systems and non-cardiac disease states [9, 30]. Notably, the C-terminus of the PP2A catalytic subunit interacts with an interface of the A and B subunits. This C-terminal region is altered by both phosphorylation and methylation that alters recruitment and docking of the regulatory subunit with the A and C subunits to form the active holoenzyme (
The phosphorylation status of the catalytic subunit of PP2A at residue Y307, a site liked with inactivation of the phosphatase by pp60vSrc, pp561ck, epidermal growth factor, and insulin receptor signaling at baseline in heart, and in heart disease was first examined [31]. Notably, phosphorylated PP2A/C Y307 was observed at baseline in human heart (
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Further, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the appended claims Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention described herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application claims benefit of U.S. Provisional Application No. 61/672,990, filed Jul. 18, 2012 and is hereby incorporated herein by reference in its entirety.
This invention was made with government support under Grant Nos. HL084583, HL083422, HL079031, HL62494, HL70250, HL089836, HL096805 awarded by the National Institutes of Health, Grant No. 0750891 awarded by the National Science Foundation and Grant No. W81XWH-10-1-0996 awarded by the U.S. Department of Defense. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61672990 | Jul 2012 | US |
