Patent Grant
11525161
Provided are compositions and methods for differentiating and diagnosing ischemic stroke and subgroups thereof (e.g., cardioembolic stroke, large vessel stroke, atherothrombotic stroke, lacunar stroke) from intracerebral hemorrhage.
Clinicians diagnose stroke based on patient history, neurological exams and brain imaging. This can be difficult and distinguishing ischemic stroke (IS) from hemorrhage can be challenging when imaging is unavailable in the acute setting.
Blood transcriptomes have provided insights into the immune response following human stroke and show promise as diagnostic biomarkers [1-6]. However, these studies have investigated only a proportion of the protein coding transcriptome, since they have used 3′-biased microarrays to measure blood mRNA expression [1-6]. Though these studies provided proof-of-principle, the vast complexity of the stroke transcriptome which is comprised of alternatively spliced isoforms remains unstudied in stroke. The importance of alternative splicing (AS) in stroke is supported by increasing evidence implicating AS in the pathogenesis of many diseases [7, 8].
AS is the process whereby exons from a single gene are included or excluded in the final mRNA transcript (
In one aspect, provided are methods for diagnosing cardioembolic ischemic stroke (CE IS) or a predisposition for experiencing CE IS. In some embodiments, the methods comprise determining a level of exon or splice variant usage or expression of a plurality of biomarkers in a biological sample from a patient, wherein an increase of the level of exon usage or expression compared to a control indicates that the patient has suffered or is at risk of experiencing CE IS, wherein the plurality of exons or splice variants of the biomarkers is selected from the biomarkers set forth in Table 1A, thereby diagnosing CE IS or a predisposition for experiencing CE IS.
In a further aspect, provided are methods for diagnosing large vessel ischemic stroke (LV IS) or a predisposition for experiencing LV IS. In some embodiments, the methods comprise determining a level of exon or splice variant usage or expression of a plurality of biomarkers in a biological sample from a patient, wherein an increase of the level of exon usage or expression compared to a control indicates that the patient has suffered or is at risk of experiencing LV IS, wherein the plurality of exons or splice variants of the biomarkers is selected from the biomarkers set forth in Table 1B, thereby diagnosing LV IS or a predisposition for experiencing LV IS.
In a further aspect, provided are methods for diagnosing lacunar ischemic stroke (L IS) or a predisposition for experiencing L IS. In some embodiments, the methods comprise determining a level of exon or splice variant usage or expression of a plurality of biomarkers in a biological sample from a patient, wherein an increase of the level of exon usage or expression compared to a control indicates that the patient has suffered or is at risk of experiencing L IS, wherein the plurality of exons or splice variants of the biomarkers is selected from the biomarkers set forth in Table 1C, thereby diagnosing L IS or a predisposition for experiencing L IS.
In a further aspect, provided are methods for diagnosing intracerebral hemorrhage (ICH) or a predisposition for experiencing ICH. In some embodiments, the methods comprise determining a level of exon usage or expression of a plurality of biomarkers in a biological sample from a patient, wherein an increase of the level of exon or splice variant usage or expression compared to a control indicates that the patient has suffered or is at risk of experiencing ICH, wherein the plurality of exons or splice variants of the biomarkers is selected from the biomarkers set forth in Table 1D, thereby diagnosing ICH or a predisposition for experiencing ICH.
In a further aspect, provided are methods of differentiating between ischemic stroke (IS) from intracerebral hemorrhage (ICH). In some embodiments, the methods comprise determining a level of exon or splice variant usage or expression of a plurality of biomarkers set forth in Table 1A, a plurality of biomarkers set forth in Table 1B, a plurality of biomarkers set forth in Table 1C, and a plurality of biomarkers set forth in Table 1D, in a biological sample from a patient, wherein detecting:
a) an increase of the level of exon or splice variant usage or expression of a plurality of exons of the biomarkers set forth in Table 1A compared to a control indicates that the patient has suffered or is at risk of experiencing cardioembolic ischemic stroke (CE IS);
b) an increase of the level of exon or splice variant usage or expression of a plurality of exons of the biomarkers set forth in Table 1B compared to a control indicates that the patient has suffered or is at risk of experiencing large vessel ischemic stroke (LV IS);
c) an increase of the level of exon or splice variant usage or expression of a plurality of exons of the biomarkers set forth in Table 1C compared to a control indicates that the patient has suffered or is at risk of experiencing lacunar ischemic stroke (L IS); and
d) an increase of the level of exon or splice variant usage or expression of a plurality of exons of the biomarkers set forth in Table 1D compared to a control indicates that the patient has suffered or is at risk of experiencing ICH; thereby differentiating between ischemic stroke (IS) from ICH.
With respect to embodiments of the methods, in some embodiments, the determining step is performed at 3 or more hours after a suspected ischemic stroke or ICH. In some embodiments, the determining step is performed at 3 or fewer hours after a suspected ischemic stroke or ICH. In some embodiments, the determining step is performed at 24 or fewer hours after a suspected ischemic stroke or ICH. In some embodiments, the determining step is performed at least 24 hours after a suspected ischemic stroke or ICH. In some embodiments, the level of expression of the biomarker is determined at the transcriptional level. In varying embodiments, the level of expression is determined by detecting hybridization of a nucleic acid probe to gene transcripts of the biomarkers in the biological sample. In varying embodiments, the hybridization step is performed on a nucleic acid microarray chip. In varying embodiments, the hybridization step is performed in a microfluidics assay plate. In varying embodiments, the level of expression is determined by direct RNA sequencing of gene transcripts of the biomarkers. In varying embodiments, the level of expression is determined by amplification of gene transcripts of the biomarkers. In varying embodiments, the amplification reaction is a polymerase chain reaction (PCR). In varying embodiments, the amplification reaction comprises quantitative reverse transcription polymerase chain reaction (qRT-PCR). In varying embodiments, the amplification reaction comprises reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) (RT-LDR/spFRET). In varying embodiments, the level of expression of the biomarker isoform is determined at the protein level. In varying embodiments, the level of expression of at least 15 biomarkers, e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 biomarkers, or all listed biomarkers in the identified Table (e.g., Table 1A, Table 1B, Table 1C, Table 1D and/or Table 1E), are determined. In varying embodiments, the methods further comprise the step of obtaining a biological sample. In varying embodiments, the biological sample is blood, serum or plasma. In varying embodiments, the increased level of exon usage of a biomarker is at least about 1.2-fold in comparison to a control. In varying embodiments, the control is the expression level of the same biomarker in an individual with no history of stroke, heart attack, or peripheral vascular disease. In varying embodiments, the control is a threshold level of expression representative of a population of individuals with no history of stroke, heart attack, peripheral vascular disease. In varying embodiments, the individual or the population of individuals has at least one vascular risk factor. In varying embodiments, the control is an individual or population of individuals who have increased expression of one or more biomarkers set forth in Table 1E. In varying embodiments, the methods further comprise the step of providing a diagnosis for ischemic stroke/ICH to the patient based on the determination and identification of the level of expression of the set of ischemic stroke/ICH biomarkers. In varying embodiments, the methods further comprise the step of providing an appropriate treatment or prevention regime for ischemic stroke/ICH to the patient. In varying embodiments, the subject has experienced or is suspected of having experienced ischemic stroke or ICH. In varying embodiments, if the patient has experienced or has a predisposition to experience ischemic stroke or ICH, further comprising the step of determining the cause or risk of the ischemic stroke or ICH
In a further aspect, provided are solid supports. In varying embodiments, the solid supports comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Table 1A. In varying embodiments, the solid supports comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Table 1B. In varying embodiments, the solid supports comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Table 1C. In varying embodiments, the solid supports comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Table 1D. In varying embodiments, the solid supports comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Table 1A, Table 1B, Table 1C and/or Table 1D. In varying embodiments, the solid support is a microarray. In varying embodiments, the microarray is suitable or configured for use in a microfluidic device. In varying embodiments, the solid supports further comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Table 1E. In varying embodiments, the solid supports further comprise a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers for diagnosing one or more of cardioembolic stroke, atherothrombotic stroke, carotid stenosis, atrial fibrillation, lacunar stroke, transient ischemic attack, transient neurological events, and hemorrhagic transformation. In varying embodiments, plurality refers to at least 15 biomarkers, e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 biomarkers, or all listed biomarkers in the identified Table (e.g., Table 1A, Table 1B, Table 1C, Table 1D and/or Table 1E).
In another aspect, provided are reaction mixtures for amplifying one or more exons of a plurality (e.g., 2, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 biomarkers, or all listed biomarkers in the identified Table, e.g., Table 1A, Table 1B, Table 1C, Table 1D and/or Table 1E) of biomarkers listed in Tables 1A-E. In varying embodiments, the reaction mixture comprises one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1A. In varying embodiments, the reaction mixture comprises one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1B. In varying embodiments, the reaction mixture comprises one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1C. In varying embodiments, the reaction mixture comprises one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1D. In varying embodiments, the reaction mixture comprises one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1A, Table 1B, Table 1C and Table 1D. In varying embodiments, the reaction mixture further comprises one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1E. Further provided is a kit comprising the reaction mixtures, as described above and herein.
In a related aspect, provided are kits. In varying embodiments, the kits comprise one or more solid supports, as described herein. In varying embodiments, the kits comprise one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1A. In varying embodiments, the kits comprise one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1B. In varying embodiments, the kits comprise one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1C. In varying embodiments, the kits comprise one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1D. In varying embodiments, the kits comprise one or more primer pairs or a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1A, Table 1B, Table 1C and Table 1D. In varying embodiments, the kits further comprising a set of primers for amplifying one or more exons of a plurality of the biomarkers set forth in Table 1E.
Definitions
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Green and Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1987-2015, Wiley Interscience; Wiley Online Library at http://onlinelibrary.wiley.com/book/10.1002/0471142727/homepage/archive.htm), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are those well-known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.
“Ischemia” or “ischemic event” as used herein refers to diseases and disorders characterized by inadequate blood supply (i.e., circulation) to a local area due to blockage of the blood vessels to the area. Ischemia includes for example, strokes and transient ischemic attacks. Strokes include, e.g., ischemic stroke (including, but not limited to, cardioembolic strokes, atheroembolic or atherothrombotic strokes, i.e., strokes caused by atherosclerosis in the carotid, aorta, heart, and brain, small vessel strokes (i.e., lacunar strokes), strokes caused by diseases of the vessel wall, i.e., vasculitis, strokes caused by infection, strokes caused by hematological disorders, strokes caused by migraines, and strokes caused by medications such as hormone therapy).
The term “transient ischemic attack,” “TIA,” or “mini-stroke” interchangeably refer to a change in the blood supply to a particular area of the brain, resulting in brief neurologic dysfunction that persists, by definition, for less than 24 hours. By definition, a TIA resolves within 24 hours, but most TIA symptoms resolve within a few minutes. If symptoms persist longer, then it is categorized as a stroke. Symptoms include temporary loss of vision (typically amaurosis fugax); difficulty speaking (aphasia); weakness on one side of the body (hemiparesis); numbness or tingling (paresthesia), usually on one side of the body, and dizziness, lack of coordination or poor balance. The symptoms of a TIA usually last a few seconds to a few minutes and most symptoms disappear within 60 minutes.
Transient neurological attacks (TNA) or transient neurological events (TNE) interchangeably refer to events involving neurological symptoms typically lasting only a few minutes or hours and no more than 24 hours. TIAs are considered focal TNAs; other events—including quickly resolving amnesia, confusion, or dizziness and fainting—are considered nonfocal TNAs.
The term “small deep infarct” or “small deep infarction” or “SDI” interchangeably refer to focal infarction of the brain due to an uncertain cause, including but not limited to, cardioembolic, atheroembolic, atherosclerotic disease of the parent artery or disease of the perforating artery.
The term “lacunar stroke” or “lacune” interchangeably refer to focal infarction of the brain due to perforating branch occlusion from microatheroma or lipohyalinosis. Implicit in this definition of lacunar stroke is that the: 1) infarction is not due to cardioembolic source; 2) infarction is not due to atherosclerotic disease of parent arteries; 3) infarction occurs in regions of the brain supplied by penetrating arteries, e.g., basal ganglia, thalamus, internal capsule, corona radiata or pons; 4) lacunar stroke is oftentimes associated with the presence of hypertension, diabetes or other vascular risk factors; and 5) infarcts tend to be smaller, generally less than 50 mm in diameter. When the cause of stroke is uncertain or likely other than perforating artery disease, then the more general term—small deep infarct—is appropriate. See, e.g., Caplan, Stroke (2003) 34(3):653-9; Norrving, Pract Neurol (2008) 8:222-228; Lastilla, Clin Exp Hypertens. (2006) 28(3-4):205-15; and Arboix and Marti-Vilalta, Expert Rev Neurother. (2009) 9(2): 179-96.
The term “intracerebral hemorrhage” and “ICH” interchangeably refer to a type of stroke caused by bleeding within the brain tissue itself. Intracerebral hemorrhage occurs when a diseased blood vessel within the brain bursts, allowing blood to leak inside the brain.
An “ischemic stroke/ICH reference expression profile” refers to the pattern of expression of a set of gene exons or splice variants or isoforms (e.g., a plurality of the exons/splice variants or isoforms of the biomarkers set forth in Tables 1A, 1B, 1C, 1D and/or 1E differentially expressed (i.e., overexpressed or underexpressed) in an individual who has suffered or is at risk of experiencing ischemia (e.g., transient cerebral ischemia, transient ischemic attacks (TIA), cerebral ischemia, cardioembolic stroke, large vessel stroke, atherothrombotic stroke, lacunar stroke), intracerebral hemorrhage (ICH) relative to the expression in a control (e.g., the expression level in an individual free of an ischemic stroke/ICH event or the expression level of a stably expressed endogenous reference biomarker). A gene exon/splice variant or isoform from Tables 1A, 1B, 1C, 1D and/or 1E that is expressed at a level that is at least about 1.2-fold, e.g., at least about 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-fold, higher than the level in a control is a gene exon/splice variant or isoform overexpressed in ischemic stroke/ICH and a gene exon/splice variant or isoform from Tables 1A, 1B, 1C, 1D and/or 1E that is expressed at a level that is at least about 1.2-fold, e.g., at least about 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-fold, lower than the level in a control is a gene isoform underexpressed in ischemic stroke/ICH. Alternately, gene exons/splice variants or isoforms that are expressed at a level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the level in a control is a gene exon/splice variant or isoform overexpressed in ischemic stroke/ICH and a gene exon/splice variant or isoform that is expressed at a level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than the level in a control is a gene exon/splice variant or isoform underexpressed in ischemic stroke/ICH.
A “plurality” refers to two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000, 2000, 5000, or more (e.g., genes). In some embodiments, a plurality refers to concurrent determination of expression levels about 15-85, 20-60 or 40-50 genes, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000, 2000, 5000, or more, genes. In some embodiments, “plurality” refers to all genes listed in one or more or all tables, e.g., all genes listed in Tables 1A, 1B, 1C, 1D and/or 1E.
As used herein, the terms “treating” and “treatment” refer to delaying the onset of, retarding or reversing the progress of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition.
The term “mitigating” refers to reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease. In certain embodiments, the reduction or elimination of one or more symptoms of pathology or disease can include, but is not limited to, reduction or elimination of one or more markers that are characteristic of the pathology or disease.
The terms “subject,” “individual,” and “patient” interchangeably refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g., equine, bovine, porcine, ovine). In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other healthworker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In certain embodiments the subject may not be under the care or prescription of a physician or other healthworker.
“Sample” or “biological sample” includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood, sputum, tissue, lysed cells, brain biopsy, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
“Array” as used herein refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as “microarrays” or colloquially “chips” have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al., Science, 251:767-777 (1991). These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase synthesis methods. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261. Arrays may comprise a planar surface or may be nucleic acids or peptides on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate as described in, e.g., U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device, as described in, e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591.
The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent hybridization conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent hybridization conditions are selected to be about 5-10° C. lower than the thermal melting point for the specific sequence at a defined ionic strength Ph. The Tm is the temperature (under defined ionic strength, Ph, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent hybridization conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at Ph 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent hybridization conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
Nucleic acids that do not hybridize to each other under stringent hybridization conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, α-carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region of a ischemic stroke/ICH-associated gene (e.g., a gene set forth in Tables 1A-E), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to TIA-associated nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
1. Introduction
Provided are exon or splice variant biomarkers, and methods and tools for using such biomarkers, that can distinguish in a subject suspected of having an ischemic event between no stroke, ischemic stroke (e.g., cardioembolic stroke, large vessel stroke and lacunar stroke) and intracerebral hemorrhage.
2. Subjects Who Can Benefit from the Present Methods
Individuals who will benefit from the present methods may be exhibiting symptoms of or suspected of having experienced a neurological event, ischemic or non-ischemic. In some embodiments, the subject has experienced or is suspected of having experienced an ischemic stroke or ICH. For example, the subject may have suffered, be currently experiencing or suspected of experiencing a small deep infarct (SDI), a transient ischemic attack (TIA), intracerebral hemorrhage (ICH), an ischemic stroke, a myocardial infarction, peripheral vascular disease, or venous thromboembolism. The subject may have or have been diagnosed with cerebral vascular disease or have one or more vascular risk factors (e.g., hypertension, diabetes mellitus, hyperlipidemia, or tobacco smoking).
In some embodiments, the subject has not experienced and/or is not at risk of having an intracerebral hemorrhage. In some embodiments, the subject has not experienced and/or is not at risk of having an intracerebral hemorrhage or hemorrhagic stroke. In some embodiments, the subject has been diagnosed as having not experienced and/or not at risk of having an intracerebral hemorrhage or hemorrhagic stroke.
In some embodiments, the levels of expression of the panel of biomarkers is determined within 3 hours of a suspected ischemic stroke or ICH. In some embodiments, the levels of expression of the panel of biomarkers are determined at 3 or more hours after a suspected ischemic stroke or ICH. In some embodiments, the levels of expression of the panel of biomarkers are determined within 6, 12, 18, 24, 36, 48 hours of a suspected ischemic stroke or ICH.
In some cases, the subject is asymptomatic, but may have a risk or predisposition to experiencing ischemic stroke/ICH, e.g., based on genetics, a related disease condition, environment or lifestyle. For example, in some embodiments, the patient suffers from a chronic inflammatory condition, e.g., has an autoimmune disease (e.g., rheumatoid arthritis, Crohn's disease inflammatory bowel disease), atherosclerosis, hypertension, or diabetes. In some embodiments, the patient has high LDL-cholesterol levels or suffers from a cardiovascular disease (e.g., atherosclerosis, coronary artery disease). In some embodiments, the patient has an endocrine system disorder, a neurodegenerative disorder, a connective tissue disorder, or a skeletal and muscular disorder. Exemplary disorders associated with, related to, or causative of TIA are discussed in co-pending application Ser. No. 13/182,630 and PCT/US2011/044023, which are hereby incorporated herein by reference in their entirety for all purposes. In some embodiments, the subject has an ABCD score that is 4 or greater (see, e.g., Josephson, et al., Stroke. (2008) 39(11):3096-8; Rothwell et al., Lancet (2005) 366(9479):29-36; and Johnston, et al., Lancet. (2007) 369(9558):283-92).
In varying embodiments, the subject may be experiencing or suspected of experiencing a small deep infarct (SDI) or a lacunar stroke. Patients presenting with clinical symptoms of lacunar infarcts or diagnosed as having lacunar syndrome will also benefit from the present diagnostic gene expression profiling. Clinical symptoms of lacunar infarcts include
pure motor hemiparesis
pure sensory stroke
sensorimotor stroke
dysarthria-clumsy hand syndrome
ataxic hemiparesis
Face, arm and leg involvement are characteristic of the first three listed symptoms. A component of ataxia is also present in the last two. Patients with a lacunar syndrome typically have no aphasia, no visuospatial disturbance, no visual field defect, generally no clear disturbance of brainstem function such as pupil abnormalities and eye movement disturbances, and no decreased level of consciousness (as a direct effect rather than as a complication of the stroke) at any time after the stroke. See, Norrving, Pract Neurol (2008) 8:222-228.
3. Biomarkers Indicative of the Occurrence or Risk of Ischemic Stroke/ICH
Biomarkers useful for the prediction, diagnosis or confirmation of the occurrence of ischemic stroke (e.g., a cardioembolic stroke, a large vessel stroke, a lacunar stroke) from an intracerebral hemorrhage (ICH) are listed in Tables 1A-1E. Determination of the gene exon/splice variant or isoform expression levels of a plurality of the biomarkers of Tables 1A-1E can be performed for the prediction, diagnosis or confirmation of the occurrence of ischemic stroke (e.g., a cardioembolic stroke, a large vessel stroke, a lacunar stroke) versus an intracerebral hemorrhage in conjunction with other biomarkers known in the art for the prediction, diagnosis or confirmation of the occurrence of ischemic stroke, in conjunction with other methods known in the art for the diagnosis of ischemic stroke, in conjunction with biomarkers described herein and known in the art useful for determining the cause of ischemic stroke and/or in conjunction with methods known in the art for determining the cause of ischemic stroke. Such biomarkers are described in co-pending and co-owned U.S. Patent Publications Nos. 2015/0018234 (“BIOMARKERS FOR DIAGNOSING ISCHEMIA”); 2012/0316076 (“BIOMARKERS FOR THE DIAGNOSIS OF LACUNAR STROKE”); 2012/0065087 (“BIOMARKERS FOR DIAGNOSIS OF STROKE AND ITS CAUSES”); 2012/0015904 (“BIOMARKERS FOR DIAGNOSIS OF TRANSIENT ISCHEMIC ATTACKS”); and 2010/0197518 (“METHODS FOR DIAGNOSING ISCHEMIA”), each of which is hereby incorporated herein by reference in its entirety for all purposes.
Determination of the expression levels of a plurality of the biomarkers of Tables 1A, 1B, 1C, 1D and/or 1E can be performed for the prediction, diagnosis or confirmation of the occurrence of ischemic stroke/ICH can also be performed independently, e.g., to diagnose whether ischemic stroke and/or ICH has occurred, to distinguish between ischemic stroke and ICH, or to determine the risk that a patient may suffer an ischemic stroke and/or ICH.
As appropriate, the expression levels of at least about 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more biomarkers (e.g., gene exons/splice variants or isoforms) from Tables 1A, 1B, 1C, 1D and/or 1E are determined. In some embodiments, the expression levels of a plurality of biomarkers in Tables 1A, 1B, 1C, 1D and/or 1E are determined. In some embodiments, the expression levels of all listed biomarkers in Tables 1A, 1B, 1C, 1D and/or 1E are determined.
In some embodiments, the level of expression of biomarkers indicative of the occurrence of ischemic stroke/ICH is determined within 72 hours, for example, within 60, 48, 36, 24, 12, 6 or 3 hours of a suspected ischemic stroke or ICH.
In various embodiments, an increased expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all, ischemic stroke-associated biomarkers of Table 1A indicates that the subject has or is likely to have experienced, is or is likely to be experiencing cardioembolic ischemic stroke:
In various embodiments, an increased expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or all, ischemic stroke-associated biomarkers of Table 1B indicates that the subject has or is likely to have experienced, is or is likely to be experiencing large vessel or atherothrombotic stroke:
In various embodiments, an increased expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or all, ischemic stroke-associated biomarkers of Table 1C indicates that the subject has or is likely to have experienced, is or is likely to be experiencing lacunar stroke:
In various embodiments, an increased expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, or all, ICH-associated biomarkers of Table 1D indicates that the subject has or is likely to have experienced, is or is likely to be experiencing intracerebral hemorrhage:
In varying embodiments, the increased expression level of the gene exon or isoform is at least about 1.2-, e.g., at least about 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-fold higher than the expression level in a control.
The overexpression or the underexpression of the biomarkers are determined with reference to a control level of expression. The control level of expression can be determined using any method known in the art. For example, the control level of expression can be from a population of individuals known to not have or be at risk for ischemic stroke or ICH or can be determined with reference to a panel of stably expressed reference biomarkers. Also, threshold levels of expression can be determined based on levels of expression in predetermined populations (e.g., known to not have or be at risk for an ischemic stroke or ICH versus known to have or be at risk for ischemic stroke/ICH). Overexpression or underexpression of a plurality of biomarkers from Tables 1A, 1B, 1C and/or 1D that is at least about 1.2-fold, e.g., at least about 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, or more, in comparison to the expression levels of a plurality of stably expressed endogenous reference biomarkers, e.g., as described herein or known in the art, is correlative with or indicates that the subject has experienced or is at risk of experiencing an ischemic stroke/ICH. Overexpression or underexpression of a plurality of biomarkers from Tables 1A, 1B, 1C and/or 1D that is at least about 1.2-fold, e.g., 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, or more, in comparison to the expression level of the same biomarker in an individual or a population of individuals who have not experienced a vascular event is correlative with or indicates that the subject has experienced or is at risk of experiencing an ischemic stroke/ICH.
Biomarkers useful for the determination and diagnosis of the cause of stroke are described, e.g., in co-owned and co-pending U.S. Patent Publications Nos. 2015/0018234 (“BIOMARKERS FOR DIAGNOSING ISCHEMIA”); 2012/0316076 (“BIOMARKERS FOR THE DIAGNOSIS OF LACUNAR STROKE”); 2012/0065087 (“BIOMARKERS FOR DIAGNOSIS OF STROKE AND ITS CAUSES”); 2012/0015904 (“BIOMARKERS FOR DIAGNOSIS OF TRANSIENT ISCHEMIC ATTACKS”); and 2010/0197518 (“METHODS FOR DIAGNOSING ISCHEMIA”), each of which is hereby incorporated herein by reference in its entirety for all purposes. In addition to evaluating the expression levels of a plurality of ischemic stroke/ICH biomarkers of differential gene exon/splice variant/isoform usages, the expression levels of a plurality of biomarkers can be measured to determine whether a suspected or predicted ischemic stroke is cardioembolic, atherosclerotic or lacunar. Furthermore, the expression levels of a plurality of biomarkers can be measured to determine if the cause of stroke is due to carotid stenosis, atrial fibrillation, lacunar stroke or transient ischemic attacks. Classification of stroke subtypes is known in the art and reviewed in, e.g., in Amarenco, et al., Cerebrovasc Dis (2009) 27:493-501. Accordingly, in some embodiments, the expression levels of at least about 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 85, 100, 200, 500, 1000 or more, ischemic stroke-associated biomarkers are independently determined. In some embodiments, the expression levels of all ischemic stroke-associated biomarkers in a panel are determined.
In various embodiments, the expression levels of a plurality of ischemic stroke-associated exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience cardioembolic stroke (a.k.a., cardiac embolism, cardioembolism emboligenic heart disease). A cardioembolic stroke occurs when a thrombus (clot) dislodges from the heart, travels through the cardiovascular system and lodges in the brain, first cutting off the blood supply and then often causing a hemorrhagic bleed. In some embodiments an increased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of IRF6 (NM_006147), ZNF254 (NM_203282), GRM5 (NM_000842///NM_001143831), EXT2 (NM_000401///NM_207122), AP3S2 (NM_005829///NR_023361), PIK3C2B (NM_002646), ARHGEFS (NM_005435), COL13A1 (NM_001130103///NM_005203///NM_080798///NM_080799///NM_080800///NM_080801///NM_080802///NM_080803///NM_080804///NM_080805///NM_080806///NM_080807///NM_080808///NM_080809///NM_080810///NM_080811///NM_080812///NM_080813///NM_080814///NM_080815), PTPN20A///PTPN20B (NM_001042357///NM_001042358///NM_001042359///NM_001042360///NM_001042361///NM_001042362///NM_001042363///NM_001042364///NM_001042365///NM_001042387///NM_001042389///NM_001042390///NM_001042391///NM_001042392///NM_001042393///NM_001042394///NM_001042395///NM_001042396///NM_001042397///NM_015605), LHFP (NM_005780), BANK1 (NM_001083907///NM_001127507///NM_017935), HLA-DOA (NM_002119), EBF1 (NM_024007), TMEM19 (NM_018279), LHFP (NM_005780), FCRL1 (NM_001159397///NM_001159398///NM_052938), OOEP (NM_001080507) and LRRC37A3 (NM_199340) is correlative with or indicates that the patient has experienced or is at risk for cardioembolic stroke. In some embodiments, a decreased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of LOC284751 (NM_001025463), CD46 (NM_002389///NM_153826///NM_172350///NM_172351///NM_172352///NM_172353///NM_172354///NM_172355///NM_172356///NM_172357///NM_172358///NM_172359///NM_172360///NM_172361), ENPP2 (NM_001040092///NM_001130863///NM_006209), C19orf28 (NM_001042680///NM_021731///NM_174983), TSKS (NM_021733), CHURC1 (NM_145165), ADAMTSL4 (NM_019032///NM_025008), FLJ40125 (NM_001080401), CLEC18A (NM_001136214///NM_182619), ARHGEF12 (NM_015313), C16orf68 (NM_024109), TFDP1 (NM_007111///NR_026580) and GSTK1 (NM_001143679///NM_001143680///NM_001143681///NM_015917) is correlative with or indicates that the patient has experienced or is at risk for cardioembolic stroke.
In various embodiments, the expression levels of a plurality of ischemic stroke-associated exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience carotid stenosis. Carotid stenosis is a narrowing or constriction of the inner surface (lumen) of the carotid artery, usually caused by atherosclerosis. An inflammatory buildup of plaque can narrow the carotid artery and can be a source of embolization. Emboli break off from the plaque and travel through the circulation to blood vessels in the brain, causing ischemia that can either be temporary (e.g., a transient ischemic attack), or permanent resulting in a thromboembolic stroke (a.k.a., atherothrombosis, large-artery atherosclerosis, atherosclerosis with stenosis). In some embodiments, an increased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of NTSE (NM_002526), CLASP2 (NM_015097), GRM5 (NM_000842///NM_001143831), PROCR (NM_006404), ARHGEFS (NM_005435), AKR1C3 (NM_003739), COL13A1 (NM_001130103///NM_005203///NM_080798///NM_080799///NM_080800///NM_080801///NM_080802///NM_080803///NM_080804///NM_080805///NM_080806///NM_080807///NM_080808///NM_080809///NM_080810///NM_080811///NM_080812///NM_080813///NM_080814///NM_080815), LHFP (NM_005780), RNF7 (NM_014245///NM_183237), CYTH3 (NM_004227), EBF1 (NM_024007), RANBP10 (NM_020850), PRSS35 (NM_153362), C12orf42 (NM_001099336///NM_198521) and LOC100127980 (XM_001720119///XM_001722650) is correlative with or indicates that the patient has experienced or is at risk for carotid stenosis. In some embodiments, a decreased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of FLJ31945 (XM_001714983///XM_001716811///XM_001718431), LOC284751 (NM_001025463), LOC100271832 (NR_027097), MTBP (NM_022045), ICAM4 (NM_001039132///NM_001544///NM_022377), SHOX2 (NM_001163678///NM_003030///NM_006884), DOPEY2 (NM_005128), CMBL (NM_138809), LOC146880 (NR_026899///NR_027487), SLC20A1 (NM_005415), SLC6A19 (NM_001003841), ARHGEF12 (NM_015313), C16orf68 (NM_024109), GIPC2 (NM_017655) and LOC100144603 (NR_021492) is correlative with or indicates that the patient has experienced or is at risk for carotid stenosis.
In various embodiments, the expression levels of a plurality of ischemic stroke-associated exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience atrial fibrillation. Atrial fibrillation (AF or A-fib) is the most common cardiac arrhythmia and involves the two upper chambers (atria) of the heart fibrillating (i.e., quivering) instead of a coordinated contraction. In some instances, cardioembolic stroke can occur as a result of atrial fibrillation. Cardioembolic stroke can be a downstream result of atrial fibrillation in that stagnant blood in the fibrillating atrium can form a thrombus that then embolises to the cerebral circulation, blocking arterial blood flow and causing ischaemic injury. In some embodiments, an increased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of SMC1A (NM_006306), SNORA68 (NR_000012), GRLF1 (NM_004491), SDC4 (NM_002999), HIPK2 (NM_001113239///NM_022740///XM_001716827///XM_925800), LOC100129034 (NR_027406///XR_079577), CMTM1 (NM_052999///NM_181268///NM_181269///NM_181270///NM_181271///NM_181272///NM_181283///NM_181296) and TTC7A (NM_020458) is correlative with or indicates that the patient has experienced or is at risk for atrial fibrillation. In some embodiments, a decreased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of LRRC43 (NM_001098519///NM_152759), MIF///SLC2A11 (NM_001024938///NM_001024939///NM_002415///NM_030807), PER3 (NM_016831), PPIE (NM_006112///NM_203456///NM_203457), COL13A1 (NM_001130103///NM_005203///NM_080798///NM_080799///NM_080800///NM_080801///NM_080802///NM_080803///NM_080804///NM_080805///NM_080806///NM_080807///NM_080808///NM_080809///NM_080810///NM_080811///NM_080812///NM_080813///NM_080814///NM_080815), DUSP16 (NM_030640), BRUNOL6 (NM_052840), GPR176 (NM_007223), C6orf164 (NR_026784) and MAP3K7IP1 (NM_006116///NM_153497) is correlative with or indicates that the patient has experienced or is at risk for atrial fibrillation.
In various embodiments, the expression levels of a plurality of ischemic stroke-associated exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience transient ischemic attacks (TIA). A transient ischemic attack is a change in the blood supply to a particular area of the brain, resulting in brief neurologic dysfunction that persists, by definition, for less than 24 hours. If symptoms persist longer, then it is categorized as a stroke. In some embodiments, an increased expression level of one or more TIA-associated biomarkers selected from the group consisting of GABRB2 (NM_000813///NM_021911), ELAVL3 (NM_001420///NM_032281), COL1A1 (NM_000088), SHOX2 (NM_003030///NM_006884), TWIST1 (NM_000474), DPPA4 (NM_018189), DKFZP434P211 (NR_003714), WIT1 (NM_015855///NR_023920), SOX9 (NM_000346), DLX6 (NM_005222), ANXA3 (NM_005139), EPHA3 (NM_005233///NM_182644), SOX11 (NM_003108), SLC26A8 (NM_052961///NM_138718), CCRL1 (NM_016557///NM_178445), FREM2 (NM_207361), STOX2 (NM_020225), ZNF479 (NM_033273///XM_001714591///XM_001719979), LOC338862 (NR_038878.1), ASTN2 (NM_014010///NM_198186///NM_198187///NM_198188), FOLH1 (NM_001014986///NM_004476), SNX31 (NM_152628), KREMEN1 (NM_001039570///NM_001039571), ALS2CR11 (NM_152525), FIGN (NM_018086), RORB (NM_006914), LOC732096 (XM_001720784///XM_001725388///XR_016064), GYPA (NM_002099), ALPL (NM_000478///NM_001127501), LHX2 (NM_004789), GALNTS (NM_014568), SRD5A2L2 (NM_001010874), GALNT14 (NM_024572), OVOL2 (NM_021220), BMPR1B (NM_001203), UNCSB (NM_170744), ODZ2 (NM_001080428///NM_001122679), RASAL2 (NM_004841///NM_170692), SHOX (NM_000451///NM_006883), C19orf59 (NM_174918), ZNF114 (NM_153608), SRGAP1 (NM_020762), ELAVL2 (NM_004432), NCRNA00032 (XM 376821///XM 938938), LOC440345 (XR 015786), FLJ30375 (XM_001724993///XM_001725199///XM_001725628), TFPI (NM_001032281///NM_006287), PTGR1 (NM_012212), ROBO1 (NM_002941///NM_133631), NR2F2 (NM_021005), GRM5 (NM_000842///NM_001143831), LUM (NM_002345), FLJ39051 (NR_033839.1), COL1A2 (NM_000089), CASP5 (NM_001136109///NM_001136110///NM_001136111///NM_001136112///NM_004347//), OPCML (NM_001012393///NM_002545), TTC6 (NM_001007795), TFAP2B (NM_003221), CRISP2 (NM_001142407///NM_001142408///NM_001142417///NM_001142435///NM_003296), SOX11 (NM_003108), ANKRD30B (XM_001716904///XM_001717561///XM_001717810), SCN2A (NM_001040142///NM_001040143///NM_021007), MYNN (NM_018657), FOXA2 (NM_021784///NM_153675), DKFZP434B061 (XR_015528///XR_040812), LOC645323 (NR_015436///NR_024383///NR_024384///XR_041118///XR_041119///XR_041120), SNIP (NM_025248), LOC374491 (NR_002815), ADAM30 (NM_021794), SIX3 (NM_005413), FLJ36144 (XR_040632///XR_040633///XR_040634), CARD8 (NM_014959), RP1-127L4.6 (NM_001010859), FAM149A (NM_001006655///NM_015398), B3GAT2 (NM_080742), SPOCK3 (NM_001040159///NM_016950), ITGBL1 (NM_004791), IQGAP3 (NM_178229), C7orf45 (NM_145268), ZNF608 (NM_020747), LOC375010 (XR_041271), LRP2 (NM_004525), TGFB2 (NM_001135599///NM_003238), SHOX2 (NM_003030///NM_006884), HOXC4///HOXC6 (NM_004503///NM_014620///NM_153633///NM_153693), ELTD1 (NM_022159), FAM182B///RP13-401N8.2 (XM_001132551///XM_001133521///XM_001718365///XM_933752), LIFR (NM_001127671///NM_002310), FOLH1 (NM_001014986///NM_004476), EHF (NM_012153), NDST3 (NM_004784), BRUNOLS (NM_021938), LOC728460 (XM_001128581///XM_001129498///XM_001723364), PDE1A (NM_001003683///NM_005019), POU2AF1 (NM_006235), FAT1 (NM_005245), PCDH11X///PCDH11Y (NM_014522///NM_032967///NM_032968///NM_032969///NM_032971///NM_032972), FLJ37786 (XR_041472///XR_041473), SLC22A4 (NM_003059), DHRS13 (NM_144683), MEG3 (NR_002766///NR_003530///NR_003531), PIWIL1 (NM_004764), LOC203274 (AL117607.1///BC080605.1), LOC100133920///LOC286297///(NR_024443///XM_001714612///XM_372109///XM_933054///XM_933058), DMRT1 (NM_021951), ADM (NM_001124), VWA3B (NM_144992), GAFA3 (XM_001715321///XM_001722922///XM_001723636), HESX1 (NM_003865), ADAMDEC1 (NM_014479), CAV1 (NM_001753), LAMB4 (NM_007356), TPTE (NM_199259///NM_199260///NM_199261), PPP1R1C (NM_001080545), HPSE (NM_001098540///NM_006665), AIM2 (NM_004833), RUNDC3B (NM_001134405///NM_001134406///NM_138290), CARD16 (NM_001017534///NM_052889), FAM124A (NM_145019), MGC39584 (XR_017735///XR_017787///XR_041937), OSM (NM_020530), RFX2 (NM_000635///NM_134433), MYBPC1 (NM_002465///NM_206819///NM_206820///NM_206821), LTBR (NM_002342), C18orf2 (NM_031416///NR_023925///NR_023926///NR_023927///NR_023928), SNRPN (NM_003097///NM_022805///NM_022806///NM_022807///NM_022808///NR_001289), FLJ36031 (NM_175884), IL1B (NM_000576), TRPM1 (NM_002420), OSTCL (NM_145303), MAPK14 (NM_001315///NM_139012///NM_139013///NM_139014), KCNJ15///LOC100131955 (NM_002243///NM_170736///NM_170737///XM_001713900///XM_001715532///XM_0), FIGN (NM_018086), HNT (NM_001048209///NM_016522), S100A12 (NM_005621), CHIT1 (NM_003465), C7orf53 (NM_001134468///NM_182597), FAM13A1 (NM_001015045///NM_014883), GNAO1 (NM_020988///NM_138736), MAPK14 (NM_001315///NM_139012///NM_139013///NM_139014), FAM55D (NM_001077639///NM_017678), PRKD2 (NM_001079880///NM_001079881///NM_001079882///NM_016457), LIMK2 (NM_001031801///NM_005569///NM_016733), C18orf54 (NM_173529), IGFBP5 (NM_000599), EVI1 (NM_001105077///NM_001105078///NM_005241), PLSCR1 (NM_021105), FOXC1 (NM_001453), LOC646627 (NM_001085474), ZNF462 (NM_021224), CNTLN (NM_001114395///NM_017738), ZNF438 (NM_001143766///NM_001143767///NM_001143768///NM_001143769///NM_001143770), DEFB105A///DEFB105B (NM_001040703///NM_152250), LOC340017 (NR_026992.1), C1orf67 (NM_144989), ACSL1 (NM_001995), ADH1B (NM_000668), SLC2A14///SLC2A3 (NM_006931///NM_153449), IL1B (NM_000576), ST3GAL4 (NM_006278///XM_001714343///XM_001726541///XM_001726562), UBE2J1 (NM_016021), PNPLA3 (NM_025225) and PAPPA (NM_002581) is correlative with or indicates that the patient has experienced or is at risk for TIA. In some embodiments, a decreased expression level of one or more TIA-associated biomarkers selected from the group consisting of NBPF10///RP11-9412.2 (NM_001039703///NM_183372///XM_001722184), SFXN1 (NM_022754), SPIN3 (NM_001010862), UNC84A (NM_001130965///NM_025154), OLFM2 (NM_058164), PPM1K (NM_152542), P2RY10 (NM_014499///NM_198333), ZNF512B (NM_020713), MORF4L2 (NM_001142418///NM_001142419///NM_001142420///NM_001142421///NM_001142422), GIGYF2 (NM_001103146///NM_001103147///NM_001103148///NM_015575), ERAP2 (NM_001130140///NM_022350), SLFN13 (NM_144682), LOC401431 (XR_040272///XR_040273///XR_040274///XR_040275), MED6 (NM_005466), BAIAP2L1///LOC100128461 (NM_018842///XM_001722656///XM_001724217///XM_001724858), LNPEP (NM_005575///NM_175920), MBNL1 (NM_021038///NM_207292///NM_207293///NM_207294///NM_207295///NM_207296), NOS3 (NM_000603), MCF2L (NM_001112732///NM_024979), KIAA1659 (XM_001723799///XM_001725435///XM_001726785), SCAMPS (NM_138967), LOC648921 (XM_001715629///XM_001720571///XR_018520), ANAPC5 (NM_001137559///NM_016237), SPON1 (NM_006108), FUS (NM_004960), GPR22 (NM_005295), GAL3ST4 (NM_024637), METTL3 (NM_019852), LOC100131096 (XM_001720907///XM_001726205///XM_001726705), FAAH2 (NM_174912), SMURF2 (NM_022739), SNRPN (NM_003097///NM_022805///NM_022806///NM_022807///NM_022808///NR_001289), FBLN7 (NM_001128165///NM_153214), GLS (NM_014905), G3BP1 (NM_005754///NM_198395), RCAN3 (NM_013441), EPHX2 (NM_001979), DIP2C (NM_014974), CCDC141 (NM_173648), CLTC (NM_004859), FOSB (NM_001114171///NM_006732), CACNA1I (NM_001003406///NM_021096), UNQ6228 (XM_001725293///XM_001725359///XM_001726164), ATG9B (NM_173681), AK5 (NM_012093///NM_174858), RBM14 (NM_006328), MAN1C1 (NM_020379), HELLS (NM_018063), EDAR (NM_022336), SLC3A1 (NM_000341), ZNF519 (NM_145287), LOC100130070///LOC100130775///LOC100131787///LOC100131905///LOC100132291///LOC100132488///RPS27 (NM_001030///XM_001721002///XM_001722161///XM_001722965///XM_001723889//), ZC3H12B (NM_001010888), IQGAP2 (NM_006633), SOX8 (NM_014587), WHDC1L2 (XM_926785), TNPO1 (NM_002270///NM_153188), TNFRSF21 (NM_014452), TSHZ2 (NM_173485), DMRTC1///DMRTC1B (NM_001080851///NM_033053), GSTM1 (NM_000561///NM_146421), GSTM2 (NM_000848///NM_001142368), PNMA6A (NM_032882), CAND1 (NM_018448), CCND3 (NM_001136017///NM_001136125///NM_001136126///NM_001760), GSTM1 (NM_000561///NM_146421), and GUSBL2 (NR_003660///XR_042150///XR_042151) is correlative with or indicates that the patient has experienced or is at risk for TIA. Further biomarkers of interest are published in Zhan, et al., Neurology. (2011) 77(19):1718-24.
In various embodiments, the expression levels of a plurality of ischemic stroke-associated exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience a transient neurological event (TNE). In some embodiments, an increased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all) ischemic stroke-associated biomarkers selected from the group consisting of UBE2J1, ELAVL3, FCGR2B, BLVRA, JMJD6, DDAH2, PTRH2, CARD16, CAV1, ZNF608, NDUFB3, SLC22A4, PCMT1, CACNA1A, CASP1 and LOC100129105 indicates that the patient has suffered or is at risk of experiencing a transient neurological event (TNE). In some embodiments, an increased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or all) ischemic stroke-associated biomarkers selected from the group consisting of AIM2, C14orf101, DNAH17, UBE2J1, LOC203274, PGS1, ZEB2, DDAH2, CARD16, SPATA4, ANXA3, WIT1, FCGR2B, CACNA1A, FKBP15, N4BP2L2, HNRNPH2, ELAVL3, ZNF608, TLR10, BLVRA, SLC22A4, RAB27A, LTBR, CARD16 III CASP1, IGFBP5, CASP5, LTB, NDUFB3, SHOX2, CAV1, CNIH4, FLJ39051, CASP1, PTRH2, LOC100129105, PCMT1, CYTH4, JMJD6, DRAM1, FCGR1B indicates that the patient has suffered or is at risk of experiencing a transient neurological event (TNE). In some embodiments, an increased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or all) ischemic stroke-associated biomarkers selected from the group consisting of CARD16, IRF7, TLR6, NMU, C13orf16, TAPBP, BTC, ZBP1, HSPA6, TWIST1, PLSCR1, SAMD9L, OSTCL, C9orf66, GYPA, ADM, ANKRD22, SHOX, ZNF354A, SRGAP1, GRM5, BAGE, XRCC4, SLC37A3, OVOL2, LIFR, RASAL2, hCG_1749898, IQGAP3, HS3ST3A1, NPR3, SIX3 and HCN1 indicates that the patient has suffered or is at risk of experiencing a transient neurological event (TNE). In some embodiments, an increased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or all) ischemic stroke-associated biomarkers selected from the group consisting of UBE2J1, CARD16, LOC203274, ZNF608, CARD16///CASP1, PTRH2, ANXA3, FCGR2B, C14orf101, LOC100129105, DDAH2, RAB27A, AIM2, CASP5, HNRNPH2, RAB27A, SHOX2, CNIH4, TLR10, ZEB2, NDUFB3, CYTH4, BLVRA, FLJ39051, SLC22A4, DNAH17, SPATA4, CACNA1A, CASP1, PGS1, LTBR, FCGR1B, IGFBP5, LTB, N4BP2L2, DRAM1, WIT1, ELAVL3, FKBP15, JMJD6, CAV1 and PCMT1 indicates that the patient has suffered or is at risk of experiencing a transient neurological event (TNE). Conversely, in some embodiments, a decreased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all) ischemic stroke-associated biomarkers selected from the group consisting of UBE2J1, ELAVL3, FCGR2B, BLVRA, JMJD6, DDAH2, PTRH2, CARD16, CAV1, ZNF608, NDUFB3, SLC22A4, PCMT1, CACNA1A, CASP1 and LOC100129105 indicates that the patient has not suffered or is not at risk of experiencing a transient neurological event (TNE). In some embodiments, a decreased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or all) ischemic stroke-associated biomarkers selected from the group consisting of AIM2, C14orf101, DNAH17, UBE2J1, LOC203274, PGS1, ZEB2, DDAH2, CARD16, SPATA4, ANXA3, WIT1, FCGR2B, CACNA1A, FKBP15, N4BP2L2, HNRNPH2, ELAVL3, ZNF608, TLR10, BLVRA, SLC22A4, RAB27A, LTBR, CARD16///CASP1, IGFBP5, CASP5, LTB, NDUFB3, SHOX2, CAV1, CNIH4, FLJ39051, CASP1, PTRH2, LOC100129105, PCMT1, CYTH4, JMJD6, DRAM1, FCGR1B indicates that the patient has not suffered or is not at risk of experiencing a transient neurological event (TNE). In some embodiments, a decreased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or all) ischemic stroke-associated biomarkers selected from the group consisting of CARD16, IRF7, TLR6, NMU, C13orf16, TAPBP, BTC, ZBP1, HSPA6, TWIST1, PLSCR1, SAMD9L, OSTCL, C9orf66, GYPA, ADM, ANKRD22, SHOX, ZNF354A, SRGAP1, GRM5, BAGE, XRCC4, SLC37A3, OVOL2, LIFR, RASAL2, hCG_1749898, IQGAP3, HS3ST3A1, NPR3, SIX3 and HCN1 indicates that the patient has not suffered or is not at risk of experiencing a transient neurological event (TNE). In some embodiments, a decreased expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or all) ischemic stroke-associated biomarkers selected from the group consisting of UBE2J1, CARD16, LOC203274, ZNF608, CARD16///CASP1, UBE2J1, PTRH2, ANXA3, FCGR2B, C14orf101, LOC100129105, DDAH2, RAB27A, AIM2, CASP5, HNRNPH2, RAB27A, SHOX2, CNIH4, TLR10, ZEB2, NDUFB3, CYTH4, BLVRA, FLJ39051, SLC22A4, DNAH17, SPATA4, CACNA1A, CASP1, PGS1, LTBR, FCGR1B, IGFBP5, LTB, N4BP2L2, DRAM1, WIT1, ELAVL3, FKBP15, JMJD6, CAV1 and PCMT1 indicates that the patient has not suffered or is not at risk of experiencing a transient neurological event (TNE).
In various embodiments, the expression levels of a plurality of ischemic stroke-associated exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience lacunar stroke. In some embodiments, an increase of the expression level of one or more biomarkers selected from the group consisting of RASEF (NM_152573), CALM1 (NM_006888), TTC12 (NM_017868), CCL3///CCL3L1///CCL3L3 (NM_001001437///NM_002983///NM_021006), CCDC78 (NM_001031737), PRSS23 (NM_007173), LAIR2 (NM_002288///NM_021270), C18orf49 (AK000229.1///BC047606.1), UTS2 (NM_006786///NM_021995), LGR6 (NM_001017403///NM_001017404///NM_021636), PROCR (NM_006404), LAG3 (NM_002286), OASL (NM_003733///NM_198213), LOC100132181 (XM_001722051.1), HLA-DRB4 (NM_021983///XM_002346251), CCL2 (NM_002982), ALS2CR11 (NM_152525), SCAND2 (NR_003654///NR_004859), GBP4 (NM_052941), RUNX3 (NM_001031680///NM_004350), TSEN54 (NM_207346), UBA7 (NM_003335), FAM179A (NM_199280), TGFBR3 (NM_003243), CCDC114 (NM_144577), AKAP9 (NM_005751///NM_147185), BNC2 (NM_017637), BZRAP1 (NM_004758///NM_024418), CCL4 (NM_002984), CHST2 (NM_004267), CSF1 (NM_000757///NM_172210///NM_172211///NM_172212), ERBB2 (NM_001005862///NM_004448), GBR56 (NM_001145770///NM_001145771///NM_001145772///NM_001145773///NM_001145774), GRAMD3 (NM_001146319///NM_001146320///NM_001146321///NM_001146322///NM_023927), GRHL2 (NM_024915), GRK4 (NM_001004056///NM_001004057///NM_182982), ITIH4 (NM_002218), KIAA1618 (NM_020954), LOC147646 (XM_001134195///XM_001134326///XM_001726058), LOC150622 (NR_026832), LOC161527 (NM_002675///NM_033238///NM_033239///NM_033240///NM_033244///NM_033246), PLEKHF1 (NM_024310), PRKD2 (NM_001079880///NM_001079881///NM_001079882///NM_016457), RGNEF (NM_001080479), SESN2 (NM_031459), SLAMF7 (NM_021181), SPON2 (NM_001128325///NM_012445), STAT1 (NM_007315///NM_139266), SYNGR1 (NM_004711///NM_145731///NM_145738), TRX21 (NM_013351), TMEM67 (NM_001142301///NM_153704///NR_024522), TUBE1 (NM_016262), and ZNF827 (NM_178835), and/or a decrease of the expression level of one or more biomarkers selected from the group consisting of HLA-DQA1 (NM_002122), FLJ13773 (AK023835.1), QKI (NM_006775///NM_206853///NM_206854///NM_206855), MPZL3 (NM_198275), FAM70B (NM_182614), LOC254128 (NR_037856.1///NR_037857.1///NR_037858.1), IL8 (NM_000584), CHML (NM_001821), STX7 (NM_003569), VAPA (NM_003574///NM_194434), UGCG (NM_003358), PDXDC1 (NM_015027), LRRC8B (NM_001134476///NM_015350), STK4 (NM_006282), GTF2H2 (NM_001515), AGFG1 (NM_001135187///NM_001135188///NM_001135189///NM_004504), BTG1 (NM_001731), CFDP1 (NM_006324), CNPY2 (NM_014255), FAM105A (NM_019018), GATM (NM_001482), GTF2H2B (NM_001042490///NM_001098728///NM 001098729///NM_001515), IGHG1 (NG 001019.5///NC 000014.8), IL18RAP (NM_003853), N4BP2 (NM_018177), PHACTR1 (NM_030948), RTKN2 (NM_145307), SLC16A1 (NM_003051), SOCS1 (NM_003745), SPAG17 (NM_206996), ST6GALNAC1 (NM_018414), STK17B (NM_004226), STT3B (NM_178862), STX16 (NM_001001433///NM_001134772///NM_001134773///NM_003763), TBC1D12 (NM_015188), TRIM4 (NM_033017///NM_033091), UACA (NM_001008224///NM_018003), and WHAMML2 (NR_026589) is correlative with or indicates that the patient has experienced or is at risk for experiencing lacunar stroke.
In various embodiments, the expression levels of a plurality of exon/splice variant biomarkers are co-determined together with the expression levels of a plurality of genes useful in the determination of whether a patient has experienced or has a predisposition to experience a hemorrhagic transformation. In varying embodiments, detecting an increase of the expression level of one or more biomarkers (e.g., 1, 2, 3, 4, 5 or 6 biomarkers) selected from the group consisting of MARCH7, SMAD4, AREG and INPP5D, and/or a decrease of the expression level of one or more biomarkers selected from the group consisting of TNFSF15 and MCFD22, compared to the control level of expression indicates that the patient suffers from or is at risk of experiencing hemorrhagic transformation of ischemic stroke. In some embodiments the methods further comprise determining a level of expression of a plurality of biomarkers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or all biomarkers) selected from the group consisting of MARCH7, SMAD4, AREG, INPP5D, IRAK3, PELI1, CPD, PRKDC, GAFA3, AMACR, MKLN1, IQCK, TSC22D2, TAF8, POLH, SYTL3, EXOC6, RIF1, RNF144B, ATXN1L, TSPYL1, RAPGEF2, MTPAP, ABHD12B, LRRC18, LOC151438, NCRNA00081, TNFSF15 and MCFD22, wherein an increase of the expression level of one or more biomarkers selected from the group consisting of MARCH7, SMAD4, AREG, INPP5D, IRAK3, PELI1, CPD, PRKDC, GAFA3, AMACR, MKLN1, TSC22D2, TAF8, SYTL3, EXOC6, RIF1, RNF144B, RAPGEF2, ABHD12B, LRRC18, LOC151438 and NCRNA00081, and/or a decrease of the expression level of one or more biomarkers selected from the group consisting of TNFSF15 MCFD22, IQCK, POLH, ATXN1L, TSPYL1 and MTPAP compared to a control level of expression indicates that the patient suffers from or is at risk of experiencing hemorrhagic transformation of ischemic stroke, thereby diagnosing the occurrence of hemorrhagic transformation of ischemic stroke or the predisposition for experiencing hemorrhagic transformation of ischemic stroke.
4. Comparison to a Control Level of Expression
The expression of the ischemic stroke/ICH-associated biomarkers are compared to a control level of expression. As appropriate, the control level of expression can be the expression level of the same ischemic stroke/ICH-associated biomarker in an otherwise healthy individual (e.g., in an individual who has not experienced and/or is not at risk of experiencing ischemic stroke/ICH). In some embodiments, the control level of expression is the expression level of a plurality of stably expressed endogenous reference biomarkers, as described herein or known in the art. In some embodiments, the control level of expression is a predetermined threshold level of expression of the same ischemic stroke/ICH-associated biomarker, e.g., based on the expression level of the biomarker in a population of otherwise healthy individuals. In some embodiments, the expression level of the ischemic stroke/ICH-associated biomarker and the ischemic stroke/ICH-associated biomarker in an otherwise healthy individual are normalized to (i.e., divided by), e.g., the expression levels of a plurality of stably expressed endogenous reference biomarkers.
In varying embodiments, a subject may experience a vascular event that may be incorrectly diagnosed as ischemic stroke/ICH. In assessing a patient with a possible ischemic stroke or intracerebral hemorrhage, sometimes patients will have events that seem like ischemic stroke/ICH but are not—for example, a simple faint. Such patients will have a biomarker profile that is negative for stroke. To determine and distinguish subjects who have not experienced an ischemic stroke/ICH from subjects who have experienced an ischemic stroke or an intracerebral hemorrhage, provided herein are exon or splice variant biomarkers that identify subjects who have not experienced ischemic stroke or an intracerebral hemorrhage as distinguished from subjects who likely have experienced an ischemic stroke and intracerebral hemorrhage. These exon or splice variant biomarkers facilitate determining and distinguishing those patients who did not have an ischemic stroke or an intracerebral hemorrhage, even if presenting with one or more symptoms that might be diagnosed as ischemic stroke/ICH.
Accordingly, in varying embodiments, an increased expression level in comparison to a control of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all), ischemic stroke/ICH-associated biomarkers of Table 1E indicates that the subject has not or is unlikely to have experienced, ischemic stroke/ICH. High levels of expression of the exon or splice variant biomarkers, e.g., as compared to historical controls, indicate that the subject has not or is unlikely to have experienced, ischemic stroke/ICH. Low levels of expression of these control exon or splice variant biomarkers, e.g., as compared to historical controls, indicate that the subject has or is likely to have experienced ischemic stroke/ICH.
In some embodiments, the overexpression or underexpression of a ischemic stroke/ICH-associated biomarker is determined with reference to the expression of the same ischemic stroke/ICH-associated biomarker in an otherwise healthy individual. For example, a healthy or normal control individual has not experienced and/or is not at risk of experiencing ischemic stroke/ICH. The healthy or normal control individual generally has not experienced a vascular event (e.g., cardioembolic stroke, large vessel stroke, lacunar stroke, TIA, ischemic stroke, myocardial infarction, peripheral vascular disease, venous thromboembolism or intracerebral hemorrhage). The healthy or normal control individual generally does not have one or more vascular risk factors (e.g., hypertension, diabetes mellitus, hyperlipidemia, or tobacco smoking). As appropriate, the expression levels of the target ischemic stroke/ICH-associated biomarker in the healthy or normal control individual can be normalized (i.e., divided by) the expression levels of a plurality of stably expressed endogenous reference biomarkers.
In some embodiments, the overexpression or underexpression of a ischemic stroke/ICH-associated biomarker is determined with reference to one or more stably expressed endogenous reference biomarkers. Internal control biomarkers or endogenous reference biomarkers are expressed at the same or nearly the same expression levels in the blood of patients who have experienced ischemic stroke/ICH as compared to control patients. Target biomarkers are expressed at higher or lower levels in the blood, serum and/or plasma of patients who have experienced or are at risk of experiencing ischemic stroke/ICH. The expression levels of the target biomarker to the reference biomarker are normalized by dividing the expression level of the target biomarker to the expression levels of a plurality of stably expressed endogenous reference biomarkers. The normalized expression level of a target biomarker can be used to predict the occurrence or lack thereof of ischemic stroke/ICH, and/or the cause of the ischemic stroke/ICH.
In some embodiments, the expression level of the ischemic stroke/ICH-associated biomarker (e.g., from Tables 1A-1D and biomarkers associated with cardioembolic stroke, atherothrombotic stroke, lacunar stroke, transient ischemic attack (TIA), hemorrhagic transformation described herein) from a patient suspected of having or experiencing an ischemic and from a control patient are normalized with respect to the expression levels of a plurality of stably expressed endogenous reference biomarkers. The expression levels of the normalized expression of the ischemic stroke/ICH-associated biomarkers are compared to the expression levels of the normalized expression of the same ischemic stroke/ICH-associated biomarker in a control patient. The determined fold change in expression=normalized expression of target biomarker in the ischemic stroke/ICH patient/normalized expression of target biomarker in control patient. In varying embodiments, ischemic stroke/ICH-associated biomarker of interest (e.g., whole gene or exon/slice variant/isoform) is divided by the geometric average of the stably expressed endogenous reference biomarkers. If the normalized values are similar to historical controls (e.g., within two standard deviations), then the samples from such patients are diagnosed as indicating that the individual has not experienced and/or is not at risk of experiencing an ischemic stroke or intracerebral hemorrhage. If the expression levels of these exons are higher or lower than expected from historical controls (e.g., greater than two standard deviations), then the samples from such patients are diagnosed as indicating that the individual has experienced and/or is at risk of experiencing an ischemic stroke or intracerebral hemorrhage. In varying embodiments, software may be employed involving Support Vector Machine (SVM) or PAM signature determination and determining which side of the hyperplane subjects falls into for classification as having an ischemic stroke or an ICH. Overexpression or underexpression of the normalized ischemic stroke/ICH-associated biomarker in the ischemic stroke/ICH patient by at least about 1.2-fold, e.g., at least about 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, or more, in comparison to the expression levels of the normalized ischemic stroke/ICH-associated biomarker in a healthy control patient indicates that the patient has experienced or is at risk of experiencing an ischemic stroke/ICH.
The biomarkers of Table 1E can also be normalized in comparison to internal control biomarkers or stably expressed endogenous reference biomarkers, as described above. The control exon biomarkers in Table 1E (upregulated in subjects who have not experienced ischemic stroke/ICH) can be divided by the geometric average of the endogenous control exons. If the normalized expression levels of the biomarkers listed in Table 1E are similar to historical controls (e.g., within two standard deviations), then the samples from these patients are diagnosed as controls who have not experienced or are unlikely to experience ischemic stroke or intracerebral hemorrhage. That is, the biomarkers of Table 1E are expressed at higher levels in controls compared to patients with ischemic stroke or intracerebral hemorrhage. If the expression levels of these exons are lower than expected from historical controls (greater than two standard deviations), then the samples from these patients are diagnosed as controls who have experienced or are likely to experience ischemic stroke or intracerebral hemorrhage. Additional biomarkers (e.g., from Tables 1A-1D and biomarkers associated with cardioembolic stroke, atherothrombotic stroke, lacunar stroke, transient ischemic attack (TIA), hemorrhagic transformation described herein) can then be used to distinguish whether the patient had an ischemic stroke or an intracerebral hemorrhage. The biomarkers of Table 1E are expressed at higher levels in subjects who have not experienced ischemic stroke/ICH compared to ischemic stroke and hemorrhage patients. They are thus lower in ischemic stroke and hemorrhage patients compared to controls. The expression of the biomarkers of Table 1E can be normalized to or divided by the expression of stably expressed endogenous reference biomarkers, described herein. If low levels of expression of the biomarkers of Table 1E compared to a normalized control are detected, this indicates the subject has experienced or is at risk to experience ischemic stroke or intracerebral hemorrhage. If higher levels of expression of the biomarkers of Table 1E compared to a normalized control are detected this indicates the subject has not experienced or is not at risk to experience ischemic stroke or intracerebral hemorrhage.
In some embodiments, the control level of expression is a predetermined threshold level. The threshold level can correspond to the level of expression of the same ischemic stroke/ICH-associated biomarker in an otherwise healthy individual or a population of otherwise healthy individuals, optionally normalized to the expression levels of a plurality of stably expressed endogenous reference biomarkers. After expression levels and normalized expression levels of the ischemic stroke/ICH-associated biomarkers are determined in a representative number of otherwise healthy individuals and individuals predisposed to experiencing ischemic stroke/ICH, normal and ischemic stroke/ICH-predisposed expression levels of the ischemic stroke/ICH-associated biomarkers can be maintained in a database, allowing for determination of threshold expression levels indicative of the presence or absence of risk to experience ischemic stroke/ICH or the occurrence of ischemic stroke/ICH. If the predetermined threshold level of expression is with respect to a population of normal control patients, then overexpression or underexpression of the ischemic stroke/ICH-associated biomarker (usually normalized) in the patient by at least about 1.2-fold, e.g., at least about 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, or more, in comparison to the threshold level indicates that the patient has experienced or is at risk of experiencing ischemic stroke/ICH. If the predetermined threshold level of expression is with respect to a population of patients known to have experienced an ischemic stroke/ICH or known to be at risk for experiencing ischemic stroke/ICH, then an expression level in the patient suspected of experiencing ischemic stroke/ICH that is approximately equal to the threshold level (or overexpressed or underexpressed greater than the threshold level of expression), indicates that the patient has experienced or is at risk of experiencing an ischemic stroke/ICH.
With respect to the stably expressed endogenous reference biomarkers used for comparison, preferably, the endogenous reference biomarkers are stably expressed in blood. Exemplary endogenous reference biomarkers that find use are published, e.g., in Stamova, et al., BMC Medical Genomics (2009) 2:49. Additional endogenous reference biomarkers include without limitation, e.g., GAPDH, ACTB, B2M, HMBS, PPIB, USP7, MAPRE2, CSNK1G2, SAFB2, PRKAR2A, PI4KB, CRTC1, HADHA, MAP1LC3B, KAT5, CDC2L1///CDC2L2, GTSE1, CDC2L1///CDC2L2, TCF25, CHP, LRRC40, hCG_2003956///LYPLA2///LYPLA2P1, DAXX, UBE2NL, EIF1, KCMF1, PRKRIP1, CHMP4A, TMEM184C, TINF2, PODNL1, FBXO42, LOC441258, RRP1, C10orf104, ZDHHCS, C9orf23, LRRC45, NACC1, LOC100133445///LOC115110, PEX16. In some embodiments, the expression levels of a plurality of stably expressed endogenous reference biomarkers are determined as a control. In some embodiments, the expression levels of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or more, stably expressed endogenous reference biomarkers described herein or known in the art, are determined as a control.
In some embodiments, the expression levels of the stably expressed endogenous reference biomarkers GAPDH, ACTB, B2M, HMBS and PPIB are determined as a control. In some embodiments, the expression levels of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more, endogenous reference biomarkers selected from the group consisting of USP7, MAPRE2, CSNK1G2, SAFB2, PRKAR2A, PI4KB, CRTC1, HADHA, MAP1LC3B, KAT5, CDC2L1///CDC2L2, GTSE1, CDC2L1///CDC2L2, TCF25, CHP, LRRC40, hCG_2003956///LYPLA2///LYPLA2P1, DAXX, UBE2NL, EIF1, KCMF1, PRKRIP1, CHMP4A, TMEM184C, TINF2, PODNL1, FBXO42, LOC441258, RRP1, C10orf104, ZDHHCS, C9orf23, LRRC45, NACC1, LOC100133445///LOC115110, PEX16 are determined as a control.
5. Methods of Detecting Biomarkers Associated with Ischemic stroke/ICH
Gene expression may be measured using any method known in the art. One of skill in the art will appreciate that the means of measuring gene expression is not a critical aspect of the invention. The expression levels of the biomarkers can be detected at the transcriptional or translational (i.e., protein) level.
In some embodiments, the expression levels of the biomarkers are detected at the transcriptional level. A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see, Green and Sambrook, supra and Ausubel, supra) and may be used to detect the expression of the biomarkers set forth in Tables 1A-1E. Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g., by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention. All forms of RNA can be detected, including, e.g., message RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA).
The selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969).
Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal. The binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tij ssen, “Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20).
The probes can be labeled either directly, e.g., with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind. Thus, the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling). Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3H, 125I, 35S, 14C, or 32P-labeled probes or the like.
Other labels include, e.g., ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).
In general, a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
Most typically, the amount of RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation which does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantifying labels are well known to those of skill in the art.
In preferred embodiments, the target nucleic acid or the probe is immobilized on a solid support. Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement.
For example, in one embodiment of the invention, microarrays are used to detect the pattern of gene expression. Microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array has a reproducible pattern of a plurality of nucleic acids (e.g., a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers set forth in Tables 1A-1E) attached to a solid support. In one embodiment, the array contains a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers listed in Table 1A. In one embodiment, the array contains a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers listed in Table 1B. In one embodiment, the array contains a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers listed in Table 1C. In one embodiment, the array contains a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers listed in Table 1D. In one embodiment, the array contains a plurality of oligonucleotide probes that hybridize to a plurality of the biomarkers listed in Table 1E. In one embodiment, the array further contains a plurality of oligonucleotide probes that hybridize to a plurality of genes useful for diagnosing ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation, transient ischemic attacks, lacunar stroke, and/or hemorrhagic transformation, as described herein and/or known in the art. In various embodiments, the array further contains a plurality of stably expressed endogenous reference biomarkers. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative read-out of relative gene expression levels in ischemic stroke/ICH (e.g., correlative with or associative of ischemic stroke/ICH, or allowing the differentiation of a transient neurological event as ischemic or non-ischemic).
In some embodiments, a sample is obtained from a subject, total mRNA is isolated from the sample and is converted to labeled cRNA and then hybridized to an array. Relative transcript levels are calculated by reference to appropriate controls present on the array and in the sample. See Mahadevappa and Warrington, Nat. Biotechnol. 17, 1134-1136 (1999).
A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer arrays (VLSIPS™), available from Affymetrix, Inc. (Santa Clara, Calif.) can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759. Integrated microfluidic systems and other point-of-care diagnostic devices available in the art also find use. See, e.g., Liu and Mathies, Trends Biotechnol. (2009) 27(10):572-81 and Tothill, Semin Cell Dev Biol (2009) 20(1):55-62. Microfluidics systems for use in detecting levels of expression of a plurality of nucleic acids are commercially available, e.g., from NanoString Technologies (on the internet at nanostring.com), Applied Biosystems (Life Technologies) (appliedbiosystems.com) and Fluidigm (fluidigm.com).
Detection can be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes). One preferred example uses an antibody that recognizes DNA-RNA heteroduplexes in which the antibody is linked to an enzyme (typically by recombinant or covalent chemical bonding). The antibody is detected when the enzyme reacts with its substrate, producing a detectable product. Coutlee et al. (1989) Analytical Biochemistry 181:153-162; Bogulayski (1986) et al. J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc. Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA duplexes, including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).
In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies that are commercially or publicly available. In addition to the art referenced above, general methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art (see, e.g., Paul (3rd ed.) Fundamental Immunology Raven Press, Ltd., N.Y. (1993); Coligan, et al., Current Protocols in Immunology, Wiley Interscience (1991-2008); Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Press, N.Y. (1988); Harlow and Lane, Using Antibodies, Cold Spring Harbor Press, N.Y. (1999); Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497 (1975)). Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al. Nature 341:544-546 (1989)). Specific monoclonal and polyclonal antibodies and antisera will usually bind with a dissociation constant (KD) of at least about 0.1 μM, preferably at least about 0.01 μM or better, and most typically and preferably, 0.001 μM or better.
The nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target. For example, the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system, in particular RT-PCR or real time PCR, and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present. Alternatively, the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation. High throughput multiplex nucleic acid sequencing or “deep sequencing” to detect captured expressed biomarker genes also finds use. High throughput sequencing techniques are known in the art (e.g., 454 Sequencing on the internet at 454.com). In varying embodiments, next generation sequencing, deep sequencing or ultra deep sequencing methodologies are applied. Deep sequencing data analysis is described, e.g., in “Deep Sequencing Data Analysis (Methods in Molecular Biology),” Noam Shomron (Editor), Humana Press; 2013 edition. Next generation sequencing is described, e.g., in “Next-Generation DNA Sequencing Informatics,” Stuart M. Brown (Editor), Cold Spring Harbor Laboratory Press; 1st edition (2013); “Next-generation Sequencing: Current Technologies and Applications,” Jianping Xu (Editor), Caister Academic Press (2014); Wilhelm, et al., Nature. (2008) 453:1239-1243; Nagalakshmi, et al., Science. (2008) 320:1344-1349; and Mortazavi, et al., Nat. Methods. (2008) 5:621-628.
In varying embodiments, the biomarkers (e.g., exons or splice variants or whole genes) are detected using RNA sequencing techniques. Methodologies for direct sequencing of RNA, e.g., without an intervening step of producing cDNA are known in the art, and described for example, in Nagalakshmi, et al., Curr Protoc Mol Biol. (January 2010) Chapter 4:Unit 4.11.1-13; Wang, et al., Nat Rev Genet. (2009) 10(1):57-63; Kwok, et al, Trends Biochem Sci. (2015) 40(4):221-232; Ramsköld, et al., Methods Mol Biol. (2012) 802:259-74; Krupp, et al., Bioinformatics. (2012) 28(8):1184-5; Oudej ans, Clin Biochem. (2015) Mar. 16. pii: S0009-9120(15)00081-8; Eij a Korpelainen and Jarno Tuimala, “RNA-seq Data Analysis: A Practical Approach (Chapman & Hall/CRC Mathematical and Computational Biology,” Chapman and Hall/CRC (Sep. 19, 2014); Ernesto Picardi, “RNA Bioinformatics (Methods in Molecular Biology),” Humana Press; 2015 edition (Jan. 11, 2015); and Shashikant Kulkarni and John Pfeifer, “Clinical Genomics,” Academic Press; 1 edition (Nov. 21, 2014).
An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, preferentially human cells, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.
In other embodiments, quantitative RT-PCR is used to detect the expression of a plurality of the biomarkers set forth in one or more of Tables 1A-1E. In one embodiment, quantitative RT-PCR is used to further detect a plurality of the biomarkers useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation, transient ischemic attacks, intracerebral hemorrhage and/or hemorrhagic transformation, as described herein and known in the art. A general overview of the applicable technology can be found, for example, in A-Z of Quantitative PCR, Bustin, ed., 2004, International University Line; Quantitative PCR Protocols, Kochanowski and Reischl, eds., 1999, Humana Press; Clinical Applications of PCR, Lo, ed., 2006, Humana Press; PCR Protocols: A Guide to Methods and Applications (Innis et al. eds. (1990)) and PCR Technology: Principles and Applications for DNA Amplification (Erlich, ed. (1992)). In addition, amplification technology is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. Methods for multiplex PCR, known in the art, are applicable to the present invention.
In varying embodiments, detection is accomplished by performing reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) (RT-LDR/spFRET). Methods for performing RT-LDR/spFRET are known in the art and described, e.g., in Peng, et al., Anal Chem. 2013 Aug. 20; 85(16):7851-8.
Accordingly, in one embodiment, provided is a reaction mixture comprising a plurality of polynucleotides which specifically hybridize (e.g., primers) to a plurality of nucleic acid sequences of the genes set forth in one or more of Tables 1A-1E. In some embodiments, the invention provides a reaction mixture further comprising a plurality of polynucleotides which specifically hybridize (e.g., primers) to a plurality of nucleic acid sequences of the genes useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation, transient ischemic attacks, intracerebral hemorrhage and/or hemorrhagic transformation, as described herein and known in the art. In some embodiments, the reaction mixture is a PCR mixture, for example, a multiplex PCR mixture.
This invention relies on routine techniques in the field of recombinant genetics. Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well-known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. Basic texts disclosing the general methods of use in this invention include Green and Sambrook et al., Molecular Cloning, A Laboratory Manual (4th ed. 2012); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1987-2015, Wiley Interscience)).
For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
In some embodiments, the expression level of the biomarkers described herein are detected at the translational or protein level. Detection of proteins is well known in the art, and methods for protein detection known in the art find use. Exemplary assays for determining the expression levels of a plurality of proteins include, e.g., ELISA, flow cytometry, mass spectrometry (e.g., MALDI or SELDI), surface plasmon resonance (e.g., BiaCore), microfluidics and other biosensor technologies. See, e.g., Tothill, Semin Cell Dev Biol (2009) 20(1):55-62.
6. Providing Appropriate Treatment and Prevention Regimes to the Patient
Upon a positive determination or confirmation that a patient has experienced ischemic stroke/ICH, and a determination of the cause of the ischemic stroke/ICH, e.g., using established clinical procedures and/or the biomarkers provided herein and known in the art (e.g., employing biomarkers described in co-pending and co-owned U.S. Patent Publications Nos. 2015/0018234 (“BIOMARKERS FOR DIAGNOSING ISCHEMIA”); 2012/0316076 (“BIOMARKERS FOR THE DIAGNOSIS OF LACUNAR STROKE”); 2012/0065087 (“BIOMARKERS FOR DIAGNOSIS OF STROKE AND ITS CAUSES”); 2012/0015904 (“BIOMARKERS FOR DIAGNOSIS OF TRANSIENT ISCHEMIC ATTACKS”); and 2010/0197518 (“METHODS FOR DIAGNOSING ISCHEMIA”) for the diagnosis of ischemic stroke/ICH), the methods further provide for the step of prescribing, providing or administering a regime for the prophylaxis or treatment of ischemic stroke/ICH. By diagnosing the occurrence and/or the cause of ischemic stroke/ICH using the biomarkers described herein, a patient can rapidly receive treatment that is tailored to and appropriate for the type of ischemic stroke/ICH that has been experienced, or that the patient is at risk of experiencing.
For example, if the expression levels of the plurality of ischemic stroke/ICH-associated biomarkers indicate the occurrence or risk of ischemic stroke/ICH, a positive diagnosis of ischemic stroke/ICH can be supported or confirmed using methods known in the art. For example, the patient can be subject to MRI imaging of brain and vessels, additional blood tests, EKG, and/or echocardiogram. Patients who have experienced ischemic transient neurological events may undergo extensive evaluation of the heart, vasculature, blood and brain, and may receive stroke prevention therapy such as antiplatelet/anticoagulation, anti-hypertensive medication and lipid lowering therapy.
If the expression levels of the plurality of biomarkers indicate the occurrence or risk of an ischemic transient neurological event (e.g., transient ischemic attacks (TIA) or transient cerebral ischemia), the patient can be prescribed a regime of medications and/or life-style adjustments (e.g., diet, exercise, stress) to minimize risk factors can be recommended, including reducing blood pressure and cholesterol levels, and controlling diabetes. Several medications can be used to decrease the likelihood of a stroke after a transient ischemic attack. The medication selected will depend on the location, cause, severity and type of TIA, if TIA has occurred. For example, the patient may be prescribed a regime of an anti-platelet drug. The most frequently used anti-platelet medication is aspirin. An alternative to aspirin is the anti-platelet drug clopidogrel (Plavix). Some studies indicate that aspirin is most effective in combination with another anti-platelet drug. In some embodiments, the patient is prescribed a combination of low-dose aspirin and the anti-platelet drug dipyridamole (Aggrenox), to reduce blood clotting. Ticlopidine (Ticlid) is another anti-platelet medication that finds use to prevent or reduce the risk of stroke in patients who have experienced TIA. In some embodiments, the patient may be prescribed a regime of an anticoagulant. Exemplary anticoagulants include aspirin, heparin, warfarin, and dabigatran. Patients having a moderately or severely narrowed neck (carotid) artery, may require or benefit from carotid endarterectomy to clear carotid arteries of fatty deposits (atherosclerotic plaques) before another TIA or stroke can occur. In some embodiments, the patient may require or benefit from carotid angioplasty, or stenting.
In cases where a non-ischemic transient neurological event (TNE) is indicated, further evaluation to the cause of the non-ischemic TNE can be performed. For example, the subject can be given tests to determine if a migraine or seizure was experienced, and receive proper treatment. Patients with non-ischemic transient neurological events undergo different diagnostic evaluation and therapy, such as EEG and anti-seizure medication for seizures, and anti-migraine medication for migraines.
If the expression levels of the plurality of biomarkers indicate the occurrence or risk of cardioembolic stroke, the patient can be prescribed or administered a regime of an anticoagulant. Exemplary anticoagulants include aspirin, heparin, warfarin, and dabigatran.
If the expression levels of the plurality of biomarkers indicate the occurrence or risk of carotid stenosis, the patient can be prescribed or administered a regime of an anti-platelet drug. The most frequently used anti-platelet medication is aspirin. An alternative to aspirin is the anti-platelet drug clopidogrel (Plavix). Some studies indicate that aspirin is most effective in combination with another anti-platelet drug. In some embodiments, the patient is prescribed a combination of low-dose aspirin and the anti-platelet drug dipyridamole (Aggrenox), to reduce blood clotting. Ticlopidine (Ticlid) is another anti-platelet medication that finds use. Patients having a moderately or severely narrowed neck (carotid) artery, may require or benefit from carotid endarterectomy. This preventive surgery clears carotid arteries of fatty deposits (atherosclerotic plaques) to prevent a first or subsequent strokes. In some embodiments, the patient may require or benefit from carotid angioplasty, or stenting. Carotid angioplasty involves using a balloon-like device to open a clogged artery and placing a small wire tube (stent) into the artery to keep it open.
If the expression levels of the plurality of biomarkers indicate the occurrence or risk of atrial fibrillation, the patient can be prescribed a regime of an anti-coagulant (to prevent stroke) and/or a pharmacological agent to achieve rate control. Exemplary anticoagulants include aspirin, heparin, warfarin, and dabigatran. Exemplary rate control drugs include beta blockers (e.g., metoprolol, atenolol, bisoprolol), non-dihydropyridine calcium channel blockers (e.g., diltiazem or verapamil), and cardiac glycosides (e.g., digoxin).
If the expression levels of the plurality of ischemic stroke/ICH-associated biomarkers indicate the occurrence or risk of lacunar stroke, a positive diagnosis of lacunar stroke can be supported or confirmed using methods known in the art. For example, the patient can be subject to clinical evaluation (e.g., determination of one or more of the lacunar syndromes, including (1) Pure motor stroke/hemiparesis, (2) Ataxic hemiparesis, (3) Dysarthria/clumsy hand, (4) Pure sensory stroke, and (5) Mixed sensorimotor stroke), radiologic imaging, retinal imaging, evaluation of blood-brain barrier permeability, evidence of microhemorrhage and blood endothelial markers (e.g., (homocysteine, intercellular adhesion molecule 1 (ICAM1), thrombomodulin (TM), tissue factor (TF) and tissue factor pathway inhibitor (TFPI); Hassan, et al., Brain (2003) 126(Pt 2):424-32; and Hassan, et al., Brain. (2004) 127(Pt 1):212-9). Upon a positive diagnosis of lacunar stroke, the patient may be administered tissue plasminogen activator within three hours of experiencing ischemic stroke/ICH if the patient is without contraindications (i.e. a bleeding diathesis such as recent major surgery or cancer with brain metastases). High doses aspirin may be given within 48 hours of experiencing ischemic stroke/ICH. For long term prevention of recurrence, medical regimens may be aimed towards correcting the underlying risk factors for lacunar infarcts such as hypertension, diabetes mellitus and cigarette smoking.
If the expression levels of the plurality of ischemic stroke/ICH-associated biomarkers indicate the occurrence or risk of hemorrhagic transformation, a positive or negative diagnosis of hemorrhagic transformation of ischemic stroke can be supported or confirmed using methods known in the art. For example, the patient can be subject to MRI imaging of brain and vessels, additional blood tests, EKG, and/or echocardiogram. Patients who have experienced or who are at risk of hemorrhagic transformation of ischemic stroke may undergo extensive evaluation of the heart, vasculature, blood and brain, and may receive reduced dosages of tissue plasminogen activator (tPA), or administration of tPA may be withdrawn or discontinued. In some embodiments, patients who have experienced or who are at risk of hemorrhagic transformation of ischemic stroke may receive an interventional therapy instead of administration of tPA. In some embodiments, patients who have experienced or who are at risk of hemorrhagic transformation of ischemic stroke may receive may receive tPA co-administered with one or more pharmacological agents to prevent, inhibit, reduce and/or mitigate the symptoms of HT, e.g., minocycline, Edaravone, Fingolimide (see, Campos, et al., Stroke. (2013) 44(2):505-11), a matrix metalloproteinase (MMP) inhibitor (e.g., an inhibitor of MMP9), an anti-inflammatory agent and/or an antioxidant. Inhibitors of MMP9 that find use are known in the art and have been described, e.g., in Intl. Appl. Nos. PCT/US2010/023585, PCT/GB2003/002138, PCT/GB2003/000741, in U.S. Patent Publ. Nos. 2004/0147573, 2005/0113344, 2010/0098659, and in Tandon, et al, Bioinformation. (2011) 5(8):310-4, Tuccinardi, et al., Bioorg Med Chem. (2008) 16(16):7749-58. Patients who have not experienced or who are not at risk of hemorrhagic transformation of ischemic stroke may also undergo extensive evaluation of the heart, vasculature, blood and brain, and may receive tissue plasminogen activator (tPA).
7. Reaction Mixtures
Further provided are reaction mixtures for diagnosing ischemic stroke/ICH or a predisposition for developing ischemic stroke/ICH. In varying embodiments, the reaction mixtures comprise a plurality of nucleic acid probes or primer sets useful for the amplification of a plurality of biomarkers (e.g., 2, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 biomarkers, or all listed biomarkers in the identified Table, e.g., e.g., Table 1A, Table 1B, Table 1C, Table 1D and/or Table 1E) of the biomarkers set forth in Tables 1A-E. In one embodiment, the reaction mixtures comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1A. In one embodiment, the reaction mixtures comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1B. In one embodiment, the reaction mixtures comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1C. In one embodiment, the reaction mixtures comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1D. In one embodiment, the reaction mixtures further comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set useful for the diagnosis of ischemic stroke, cardioembolic stroke, atherothrombotic stroke, carotid stenosis, lacunar stroke, atrial fibrillation, transient ischemic attacks (TIA), transient neurological events (TNEs), intracerebral hemorrhage and/or hemorrhagic transformation, as described herein. The probes may be immobilized on an array as described herein.
In varying embodiments, the reaction mixtures further can comprise appropriate buffers, salts, polymerases, dNTPs and other reagents to facilitate amplification and/or detection reactions (e.g., primers, labels) for amplifying one or more exons of a plurality of the biomarkers set forth in Tables 1A-E.
In some embodiments, the reaction mixtures can be provided in one or more reaction vessels that have aliquots of some or all of the reaction components of the reaction mixtures in them. Aliquots can be in liquid or dried (e.g., freeze-dried) form. Reaction vessels can include sample processing cartridges or other vessels that allow for the containment, processing and/or amplification of samples in the same vessel.
Further contemplated are kits comprising the reaction mixtures described above and herein.
8. Solid Supports and Kits
The invention further provides, a solid support comprising a plurality of nucleic acid probes that hybridize to a plurality (e.g., two or more, or all) of the biomarkers set forth in Tables 1A-E, and optionally stably expressed endogenous reference biomarkers, as described herein. For example, the solid support can be a microarray attached to a plurality of nucleic acid probes that hybridize to a plurality (e.g., two or more, or all) of the biomarkers set forth in Tables 1A-E, and optionally stably expressed endogenous reference biomarkers.
In various embodiments, the solid supports are configured to exclude genes not associated with or useful to the diagnosis, prediction or confirmation of ischemic stroke or ICH. For example, oligonucleotide probes that hybridize to genes or gene exons/splice variants/isoforms that are overexpressed or underexpressed less than 1.2-fold in subjects with ischemic stroke/ICH in comparison to a control level of expression can be excluded from the present solid supports. In some embodiments, oligonucleotide probes that hybridize to genes or gene exons that are overexpressed or underexpressed less than 1.2-fold in subjects with ischemic stroke, including transient cerebral ischemia, lacunar stroke, cardioembolic stroke, atherothrombotic stroke, TIA, TNE, carotid stenosis, and stroke subsequent to atrial fibrillation, in comparison to a control level of expression can be excluded from the present solid supports.
In various embodiments, the solid supports are configured to include only oligonucleotide probes that hybridize to genes or gene exons associated with or useful to the diagnosis, prediction or confirmation of ischemic stroke and/or ICH. For example, in some embodiments, only oligonucleotide probes that hybridize to genes or gene exons that are overexpressed or underexpressed more than 1.2-fold in subjects with ischemic stroke/ICH in comparison to a control level of expression are included in the present solid supports. In some embodiments, only oligonucleotide probes that hybridize to genes or gene exons that are overexpressed or underexpressed more than 1.2-fold in subjects with ICH or ischemic stroke (e.g., including transient cerebral ischemia, lacunar stroke, cardioembolic stroke, atherothrombotic stroke, TIA, TNE, carotid stenosis and stroke subsequent to atrial fibrillation), in comparison to a control level of expression are included in the present solid supports.
The solid support may optionally further comprise a plurality of oligonucleotide probes that hybridize to a plurality (e.g., two or more, or all) of the biomarkers useful for the diagnosis of ICH/ischemic stroke (e.g., cardioembolic stroke, carotid stenosis, and/or atrial fibrillation), as described herein. In various embodiments, the solid support comprises 5000, 4000, 3000, 2000, 1000 or fewer (e.g., 900, 800, 700, 600, 500 or fewer) nucleic acid probes that hybridize to a plurality of ischemic stroke/ICH-associated genes, as described herein. The solid support may be a component in a kit.
The invention also provides kits for diagnosing ischemic stroke/ICH or a predisposition for developing ischemic stroke/ICH. For example, the invention provides kits that include one or more reaction vessels that have aliquots of some or all of the reaction components of the reaction mixtures in them. Aliquots can be in liquid or dried form. Reaction vessels can include sample processing cartridges or other vessels that allow for the containment, processing and/or amplification of samples in the same vessel. The kits may comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality (e.g., two or more, or all) of the biomarkers set forth in Tables 1A-E. In one embodiment, the kits comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1A. In one embodiment, the kits comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1B. In one embodiment, the kits comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1C. In one embodiment, the kits comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set forth in Table 1D. In one embodiment, the kits further comprise a plurality of nucleic acid probes or primer sets that hybridize to a plurality of the biomarkers set useful for the diagnosis of ischemic stroke, cardioembolic stroke, atherothrombotic stroke, carotid stenosis, lacunar stroke, atrial fibrillation, transient ischemic attacks (TIA), transient neurological events (TNEs), intracerebral hemorrhage and/or hemorrhagic transformation, as described herein. The probes may be immobilized on an array as described herein.
In addition, the kit can comprise appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers, labels) for determining the expression levels of a plurality of the biomarkers set forth in Tables 1A-E. In one embodiment, the kit comprises appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers, labels) for determining the expression levels of a plurality of the biomarkers set forth in Table 1A. In one embodiment, the kit comprises appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers) for determining the expression levels of a plurality of the biomarkers set forth in Table 1B. In one embodiment, the kit comprises appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers, labels) for determining the expression levels of a plurality of the biomarkers set forth in Table 1C. In one embodiment, the kit comprises appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers) for determining the expression levels of a plurality of the biomarkers set forth in Table 1D. In one embodiment, the kit further comprises appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers) for determining the expression levels of a plurality of the biomarkers useful for the diagnosis of ischemic stroke, cardioembolic stroke, atherothrombotic stroke, lacunar stroke, carotid stenosis, atrial fibrillation, and/or transient ischemic attacks (TIA), transient neurological events (TNEs) as described herein. The kits can also include written instructions for the use of the kit.
In one embodiment, the kits comprise a plurality of antibodies that bind to a plurality of the biomarkers set forth in Tables 1A-E. The kits may further comprise a plurality of antibodies that bind to a plurality of the biomarkers useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation, transient ischemic attacks (TIA), transient neurological events (TNEs), intracerebral hemorrhage and/or hemorrhagic transformation, as described herein. The antibodies may or may not be immobilized on a solid support, e.g., an ELISA plate.
The following examples are offered to illustrate, but not to limit the claimed invention.
Abstract
Background: Whole transcriptome studies have used 3′-biased expression microarrays to study genes regulated in blood of ischemic stroke patients. However, alternatively spliced messenger RNA isoforms have not been investigated for ischemic stroke or intracerebral hemorrhage (ICH) in animals or humans. Alternative splicing is the mechanism whereby a single gene's exons combine to produce distinct mRNA and protein isoforms. RNA-sequencing (RNA-Seq) was used to determine if alternative splicing varies for ICH and different ischemic stroke causes (cardioembolic, large vessel, lacunar) as compared to each other and controls.
Methods: Paired-end RNA-Seq was performed using Illumina Solexa technology to a depth of 200×106reads for splicing analysis on twenty whole-blood samples. Differential alternative splicing was assessed using 1-way-ANOVA (FDR p<0.05). Differential exon-usage was calculated between each group (p<0.0005; (fold change|>1.2).
Results: 412 genes displayed differential alternative splicing among the groups. They were involved in cellular immune response, cell death and cell survival pathways implicated in stroke. Distinct expression signatures based on usage of 308 exons (292 genes) differentiated the groups.
Conclusions: This pilot study demonstrates alternatively spliced genes from whole blood differ in ICH compared to ischemic stroke, and differ between different ischemic stroke etiologies.
Methods
Stroke patients and control subjects were randomly selected from all those recruited at the UC Davis Medical Center between 2008 and 2012. Stroke patients were chosen to represent the major ischemic stroke etiologies (cardioembolic, large vessel, lacunar) or had intracerebral hemorrhages (ICH). Control subjects were selected to match the stroke subjects by age, race and sex, to have vascular risk factors and no cardiovascular events. All subjects provided Informed Consent. The UCD Institutional Review Board approved this study. IS diagnosis and causes were assessed as previously described [5, 9]. ICH patients had deep ICH confirmed by CT and/or MRI brain scans, and were associated with hypertension without evidence of vascular malformation, tumor or aneurysm. Controls had vascular risk factors without evidence of stroke. Blood was drawn into PAXgene tubes at 5.8 to 101.2 h following IS or ICH. RNA from whole blood was isolated as previously described [3].
Whole blood RNA was used to prepare mRNA libraries using the TruSeq RNA Sample Prep v2 kit and protocol (Illumina). Paired-end 100 bp RNA-Seq reads on whole blood RNA were obtained by Illumina Solexa sequencing by synthesis on Illumina HiSeq2000 to a depth of 200×106 reads. Bowtie 2 was used to map reads to a reference genome (Hg19) and generate bam files for analysis [10]. RNA transcript quantification was performed using Hg19 AceView transcripts in Partek Genomics Suite 6.6 RNA-Seq workflow. DAS was determined with ANOVA on Group (FDR p<0.05), and differential exon-usage was assessed between each two groups (p<0.0005, |fold change|>|1.2|).
Principal Component Analysis (PCA) and Hierarchical Clustering were performed in Partek. Ingenuity Pathway Analysis (IPA®) and DAVID identified regulated pathways and processes as described previously [6].
Results
Subject Demographics. Subject demographics and clinical characteristics are presented in Table 2. Only Caucasian males were studied because of the small group sizes. Demographics for age, time since event for IS or ICH, and vascular risk factors were not significantly different. Coverage of a wide range of post-stroke biology was obtained by selecting patients with early (5.8 hours) through late (101.2 hours) blood draw times after stroke event. Although this time range seems wide, means are similar between stroke groups. Cardioembolic post-event blood draw times were, on average, 33.7 hours; large vessel averaged 47.4 hours; lacunar averaged 34.6 hours; and ICH averaged 29.4 hours (Table 2).
RNA Sequencing Alignments. RNA sequencing alignments statistics for all samples among the five groups are presented in Table 3. Cardioembolic stroke samples had on average, 1.60E+08 alignments; large vessel had 1.65E+08 alignments; lacunar stroke had 1.64E+08 alignments; and ICH and control each averaged 1.59E+08 alignments.
Distinct Alternatively Spliced Transcriptomes from Whole Blood of Patients with ICH, Different IS Etiologies and Controls. 412 genes display DAS in the whole blood transcriptomes of patients with IS (cardioembolic, large vessel, and lacunar), ICH and controls (FDR p<0.05; Table 4). These genes are involved in cellular immunity, cytokine signaling and cell death and survival pathways (Table 5). Pathways with higher over-representation of DAS genes between groups include: CD28 signaling in T helper cells, CDC42 signaling, Nur77 signaling in T lymphocytes, fMLP signaling in neutrophils, and interferon signaling (Table 5). Molecular and cellular functions best representing the DAS genes are: cell death/survival of immune cells/leukocytes, cell-to-cell signaling and interaction, including activation, recruitment and adhesion of leukocytes, antigen presenting cells, activation of T lymphocytes, adhesion of vascular endothelial cells and immune response of neutrophils (Table 6).
Specific Exon-Usage Profiles for IS and ICH. Distinct differential expression signatures based on 308 exons (292 genes) (p<0.0005, fold change>|1.2|; Table 1) separated the three causes of IS, as well as ICH and controls (PCA
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Discussion
Although DAS is implicated in many human diseases, this is the first study to report DAS for ICH, IS and controls, and is the first to show that DAS is different for different causes of IS. Identification of specific DAS for different stroke etiologies suggests the immune response varies for each condition. This will likely be important for understanding pathogenesis of each stroke cause and biomarker development.
This study identified several pathways, molecular functions and genes previously reported in human IS using 3′-biased microarrays [6, 11]. These include: actin cytoskeleton signaling, CCR5 signaling in macrophages, NF-κB activation, α-adrenergic signaling, cellular growth and proliferation, cell death and survival, cell morphology, hematopoiesis, hematological system development and function and inflammatory response/disease [4, 5, 12, 13]. This study's small sample sizes preclude detailed interpretations of biological pathways. However, these pilot data suggest DAS involvement in IS and ICH pathophysiology which varies in ICH and in different IS etiologies.
Results suggest DAS differs in blood leukocytes following different cerebrovascular events. Due to the pilot nature of the study, and the lack of human transcriptome data following ICH, we will discuss only a few of the genes with differential exon usage in ICH. Among the genes that differentiated ICH were INPP5D (inositol polyphosphate-5-phosphatase) and ITA4 (integrin alpha 4). INPP5D is a regulator of myeloid cell proliferation and programming and was previously identified as correlating with increased tendency to hemorrhagic transformation of ischemic stroke [14]. ITA4 is involved in leukocyte recruitment [15] and leukocytes are intimately associated with IS and ICH [11]. For example, leukocytes are involved in clotting, and interact with injured vessels and brain following ICH and IS [11]. In addition, vascular endothelial growth factor (VEGF) signaling, which predisposes to hemorrhage because of new vessel formation [16], was implicated in ICH by several DAS genes, including NAV1 (neuron navigator 1), PDGFC (platelet derived growth factor C) and CCM2 (cerebral cavernous malformation 2). CCM2 mutations cause cerebral cavernous malformations leading to a predisposition for abnormal vessels and cerebral hemorrhage [17]. Interestingly, exosomes may be involved in ICH as evidenced by the differential expression of EXOSC1 (exosome component 1) and EXOSC9 (exosome component 9), coding for core components of the exosome complex [18]. Although exosomes have been implicated in neuroinflammation, neurodegeneration and cancer, they have not previously been associated with ICH [19, 20]. Finally, DGCR8 (microprocessor complex subunit) is involved in the biogenesis of microRNAs [21], thus suggesting an interplay between alternative splicing and miRNA in ICH.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application is the U.S. national phase under 35 U.S.C. § 371 of Intl. Appl. No. PCT/US2016/031028, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/159,502, filed on May 11, 2015, which are hereby incorporated herein by reference in their entireties for all purposes.
This invention was made with Government support under Grant Nos. NS075035, NS079153 and AG042292, all awarded by the National Institutes of Health. The government has certain rights in this invention.
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