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US20120316076A1 - Biomarkers for the diagnosis of lacunar stroke - Google Patents

Biomarkers for the diagnosis of lacunar stroke Download PDF

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US20120316076A1
US20120316076A1 US13/410,025 US201213410025A US2012316076A1 US 20120316076 A1 US20120316076 A1 US 20120316076A1 US 201213410025 A US201213410025 A US 201213410025A US 2012316076 A1 US2012316076 A1 US 2012316076A1
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stroke
lacunar
expression
biomarkers
lacunar stroke
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Frank Sharp
Glen C. JICKLING
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University of California San Diego UCSD
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Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SHARP, FRANK, JICKLING, GLEN C.
Publication of US20120316076A1 publication Critical patent/US20120316076A1/en
Priority to US15/091,181 priority patent/US10196690B2/en
Priority to US16/228,030 priority patent/US11136626B2/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Definitions

  • the present invention relates to expression profiling to differentiate stroke of lacunar etiology from non-lacunar stroke.
  • SDI Small deep infarcts
  • lacunar stroke account for greater than one quarter of all ischemic strokes.
  • SDI cause the smallest amount of brain injury of all stroke subtypes, long-term outcomes are significant with 42% of lacunar stroke patients being dependent by 3 years (Samuelsson et al., Stroke (1996) 27 (5):842-6; Lee, et al., Int J Cardiol . (2009) March 25; Giroud, et al, Rev Neurol (Paris). (1991) 147 (8-9):566-72; Clavier, et al., Stroke . (1994) 25 (10):2005-9; Carod-Artal, et al., Stroke .
  • lacunar strokes are indicative of cardiovascular disease with an annual death rate of 2.8% and an increased risk of recurrent stroke, white matter disease and cognitive impairment (Samuelsson, et al., supra; Norrving, Lancet Neurol . (2003) 2 (4):238-45; Jackson, et al., Brain . (2005) 128 (Pt 11):2507-17).
  • lacune was first used to describe small subcortical infarctions in the 1800s by Dechambre and Durand-Fardel.
  • Miller Fisher described the lacunar hypothesis, correlating the clinical symptoms of lacunar syndromes with pathologic findings of single perforating branch occlusion from microatheroma or lipohyalinosis (Fisher, Acta Neuropathol . (1968) 12 (1):1-15; Fisher, Neurology (1965) 15:774-84; Fisher, Neurology (1982) 32 (8):871-6; and Bamford and Warlow, Stroke . (1988) 19 (9):1074-82).
  • the lacunar hypothesis distinguishes lacunar stroke from other causes of SDI, including disease of the parent artery and embolism of arterial or cardiac origin. Determining whether an SDI is of small vessel lacunar or non-small vessel etiology remains a topic of controversy and investigation (Millikan, et al., Stroke (1990) 21 (9):1251-7; Futrell, Stroke (2004) 35 (7):1778-9; Norrving, Stroke (2004) 35 (7):1779-80; Davis and Donnan, Stroke (2004) 35 (7):1780-1; and Maron, et al., J Am Coll Cardiol . (2002) 39 (2):301-7).
  • An embolic cause of stroke warrants a different investigative strategy and treatment than other ischemic stroke syndromes.
  • it is important to diagnose disease that would change management, such as carotid surgery for symptomatic carotid stenosis and warfarin for symptomatic atrial fibrillation. Therefore, ascertaining the etiology of SDI is not only of academic interest but also of clinical significance.
  • lacunar syndromes can be mimicked by non-lacunar disease, such as cortical infarction, hemorrhagic stroke and non-vascular disease (Wessels, et al., Stroke (2005) 36 (4):757-61; Arboix, et al., BMC Neurol . (2010) 10:31).
  • non-lacunar disease such as cortical infarction, hemorrhagic stroke and non-vascular disease
  • infarction in the regions of the penetrating arteries (basal ganglia, thalamus, internal capsule, corona radiata and pons) can result from non-lacunar disease, including disease of the parent artery and emboli of arterial or cardiac origin.
  • Infarct diameter ⁇ 15 mm is also predictive of lacunar stroke, since this is the approximate vascular territory of a single penetrating artery (Bang, et al., Cerebrovasc Dis . (2007) 24 (6):520-9; Cho, et al., Cerebrovasc Dis . (2007) 23 (1):14-9; and Lodder, Cerebrovasc Dis . (2007) 24 (1):156-7).
  • a stroke is of lacunar or non-lacunar etiology.
  • the present invention is based, in part, on using gene expression profiling to distinguish patients who have suffered or are at risk of suffering lacunar stroke from patients who have suffered or are at risk of suffering embolic strokes using a gene expression profiling.
  • the gene expression profiles further find use to predict the cause of stroke in SDI of unclear cause (SDI size >15 mm or SDI with potential embolic source). It has recently been demonstrated that cardioembolic and large vessel causes of stroke have unique gene expression signatures (Jickling, et al., Ann Neurol . (2010) 68 (5):681-92; and Xu, et al., J Cereb Blood Flow Metab . (2008) 28 (7):1320-8).
  • signatures can be used to categorize, diagnose and treat stroke patients by cause based on a profile of differentially expressed genes.
  • the identified genes were predominantly expressed in inflammatory cells associated with each stroke subtype.
  • the present invention is based, in part, on the identification of a profile of differentially expressed genes useful to distinguish lacunar stroke from non-lacunar stroke and to predict etiology in SDI of unclear cause.
  • the present invention provides biomarker useful for diagnosing the occurrence or risk of lacunar stroke and for distinguishing the occurrence or risk of lacunar stroke from non-lacunar stroke. Accordingly, in one aspect, the invention provides methods for diagnosing the occurrence of lacunar stroke or a predisposition for experiencing lacunar stroke. In some embodiments, the methods comprise:
  • the invention provides methods for distinguishing the occurrence of lacunar stroke or a predisposition for experiencing lacunar stroke from the occurrence of non-lacunar stroke or a predisposition for experiencing non-lacunar stroke.
  • the methods comprise:
  • the invention provides methods for diagnosing lacunar stroke or a predisposition for developing lacunar stroke.
  • the methods comprise determining a level of expression of a plurality of lacunar stroke-associated biomarkers in a biological sample from a patient, wherein an increase or decrease of the level compared to a control level is correlative with or indicates that the patient suffers from or is at risk of developing lacunar stroke;
  • a decrease of the expression level of one or more biomarkers selected from the group consisting of RASEF, CALM1, TTC12, CCL3, CCL3L1, CCL3L3, CCDC78, PRSS23, and LAIR2, and an increase of the expression level of one or more biomarkers selected from the group consisting of HLA-DQA1, FLJ13773, and QKI, compared to the expression level of the plurality of endogenous reference biomarkers is correlative with or indicates that the patient suffers from or is at risk of experiencing non-lacunar stroke.
  • the expression levels of the biomarkers are concurrently or sequentially determined.
  • the methods further comprise the step of obtaining a biological sample from the patient.
  • the biological sample is blood, serum or plasma.
  • the method is performed in a clinical laboratory. In some embodiments, the method is performed at the point of care.
  • the plurality of stably expressed endogenous reference biomarkers are selected from USP7, MAPRE2, CSNK1G2, SAFB2, PRKAR2A, PI4KB, CRTC1, HADHA, MAP1LC3B, KAT5, GTSE1, CDC2L1///CDC2L2, TCF25, CHP, LRRC40, hCG — 2003956///LYPLA2///LYPLA2P1, DAXX, UBE2NL, EIF1, KCMF1, PRKRIP1, CHMP4A, TMEM184C, TINF2, PODNL1, FBXO42, LOC441258, RRP1, C10orf104, ZDHHC5, C9orf23, LRRC45, NACC1, LOC100133445///LOC115110 and PEX16.
  • the lacunar stroke-associated biomarkers are overexpressed or underexpressed at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, or more, in comparison to the expression levels of a plurality of stably expressed endogenous reference biomarkers.
  • the level of expression of about 15-85, 20-70, 30-60 or 40-50 lacunar stroke-associated biomarkers are determined. In some embodiments, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 lacunar stroke-associated biomarkers are determined. In some embodiments, the expression levels of at least about 3, 5, 10, 15, 20, 25, 30 or more lacunar stroke-associated biomarkers from Table 3 are determined. In some embodiments, the expression levels of at least about 3, 5, 10, 15, 20, 25, 30 or more lacunar stroke-associated biomarkers from Table 4 are determined.
  • the determining step is performed within 72 hours, for example, within 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours or 3 hours, after a suspected ischemic event.
  • the patient is asymptomatic. In some embodiments, the patient is exhibiting symptoms of ischemic stroke, e.g., of having experienced an ischemic event, of experiencing an ischemic event, or of an imminent ischemic event. In some embodiments, the patient has suffered an ischemic event. In some embodiments, the determining step is performed at 3 or fewer hours after the ischemic event. In some embodiments, the determining step is performed 3 or more hours after the ischemic event.
  • the patient has at least one vascular risk factor. In some embodiments, the patient has experienced a small deep infarction (SDI). In some embodiments, the patient shows evidence of microhemorrhage. In some embodiments, the patient is non-Caucasian. In some embodiments, the patient does not have arterial disease ipsilateral to the stroke.
  • SDI small deep infarction
  • the patient shows evidence of microhemorrhage. In some embodiments, the patient is non-Caucasian. In some embodiments, the patient does not have arterial disease ipsilateral to the stroke.
  • the methods are computer implemented. Such computer-implemented methods may also provide an output of the comparison of expression levels.
  • SDI small deep infarction
  • the level of expression of the biomarker is determined at the transcriptional level. In some embodiments, the level of expression is determined by detecting hybridization of a lacunar stroke-associated gene probe to gene transcripts of the biomarkers in the biological sample.
  • the methods further comprise the step of performing additional diagnostic tests useful for identifying whether a patient has experienced or has a predisposition to experience lacunar stroke, e.g., based on imaging or ultrasound techniques.
  • the methods further comprise performing one or more diagnostic tests selected from the group consisting of X-ray computed tomography (CT), magnetic resonance imaging (MRI) brain scanning, vascular imaging of the head and neck with doppler or magnetic resonance angiography (MRA), CT angiography (CTA), electrocardiogram (e.g., EKG or ECG), cardiac ultrasound and cardiac monitoring.
  • CT computed tomography
  • MRA magnetic resonance imaging
  • CTA CT angiography
  • electrocardiogram e.g., EKG or ECG
  • cardiac ultrasound and cardiac monitoring e.g., EKG or ECG
  • the patient is subjected to cardiac monitoring for at least 2 days, e.g., for 2-30 days or for 7-21 days, e.g., for 2, 5, 7, 10, 12, 14, 18, 20, 21, 25, 28, 30, or more days, as appropriate.
  • the location of the infarction is determined.
  • An infarction located in a subcortical region of the brain is associated with or correlated with a diagnosis of lacunar stroke.
  • An infarction located in a cortical region of the brain e.g., in regions of the penetrating arteries, e.g., basal ganglia, thalamus, internal capsule, corona radiata and/or pons, is associated with or correlated with a diagnosis of non-lacunar stroke.
  • the size of the infarction is determined.
  • the methods further comprise the step of recommending or providing a regime of treatment to the patient appropriate to the determined cause of stroke. For example, in patients diagnosed as experiencing or having a predisposition for experiencing lacunar stroke, the methods further provide for recommending or providing a regime of treatment or prevention for lacunar stroke.
  • the methods may further comprise the step of determining the cause or risk of ischemic stroke if the patient has experienced or has a predisposition to experience non-lacunar stroke.
  • the methods may further comprise the step of recommending or providing a regime of treatment to the patient appropriate to the determined cause of non-lacunar stroke. For example, in patients diagnosed as experiencing or having a predisposition for experiencing cardioembolic stroke, the methods further provide for recommending or providing a regime of treatment or prevention for cardioembolic stroke. In patients diagnosed as experiencing or having a predisposition for experiencing carotid stenosis, the methods further provide for recommending or providing a regime of treatment or prevention for carotid stenosis.
  • the methods further provide for recommending or providing a regime of treatment or prevention for atrial fibrillation.
  • the methods further provide for recommending or providing a regime of treatment or prevention for transient ischemic attack.
  • the level of expression of the biomarker is determined at the transcriptional level.
  • the level of expression is determined by detecting hybridization of an ischemic stroke-associated gene probe to gene transcripts of the biomarkers in the biological sample.
  • the hybridization step is performed on a nucleic acid array chip.
  • the hybridization step is performed in a microfluidics assay plate.
  • the level of expression is determined by amplification of gene transcripts of the biomarkers.
  • the amplification reaction is a polymerase chain reaction (PCR).
  • the level of expression of the biomarker is determined at the protein level.
  • the invention provides a solid support comprising a plurality of nucleic acids that hybridize to a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, LAIR2, C18orf49, MPZL3, UTS2, FAM70B, UTS2, LOC254128, LGR6, IL8, CHML, STX7, PROCR, VAPA, LAG3, OASL, LOC100132181, HLA-DRB4, CCL2, UGCG, PDXDC1, ALS2CR11, SCAND2, GBP4, RUNX3, LRRC8B, TSEN54, UBA7, STK4, FAM179A, TGFBR3, CCDC114, GTF2H2, AKAP9, BNC2, BZRAP1, CCL4, CHST2, CSF1, ERBB2, G
  • the solid support may comprise, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more, nucleic acids that hybridize to a plurality of lacunar stroke-associated genes.
  • the solid support may be provided in a kit.
  • the solid support comprises a plurality of nucleic acids that hybridize to a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, LAIR2, C18orf49, MPZL3, UTS2, FAM70B, UTS2, LOC254128, LGR6, IL8, CHML, STX7, PROCR, VAPA, LAG3, OASL, LOC100132181, HLA-DRB4, CCL2, UGCG, PDXDC1, ALS2CR11, SCAND2, GBP4, RUNX3, LRRC8B, TSEN54, UBA7, STK4, FAM179A, TGFBR3, CCDC114 and GTF2H2.
  • a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CA
  • the solid support comprises a plurality of nucleic acids that hybridize to a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, LAIR2, C18orf49, MPZL3, UTS2, FAM70B, UTS2, LOC254128, LGR6, IL8, CHML, STX7, PROCR, VAPA, LAG3, OASL, LOC100132181, HLA-DRB4, CCL2, UGCG, PDXDC1, and ALS2CR11.
  • a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, LAIR2, C18orf49, MPZ
  • the solid support comprises a plurality of nucleic acids that hybridize to a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, LAIR2, C18orf49, MPZL3, UTS2, FAM70B, UTS2, LOC254128, LGR6, IL8, CHML, and STX7.
  • a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, LAIR2, C18orf49, MPZL3, UTS2, FAM70B, UTS2, LOC254128, LGR6, IL8, CHML, and STX7.
  • the solid support comprises a plurality of nucleic acids that hybridize to a plurality of lacunar stroke-associated genes selected from the group consisting of HLA-DQA1, FLJ13773, RASEF, CALM1, QKI, TTC12, CCL3///CCL3L1///CCL3L3, CCDC78, PRSS23, and LAIR2.
  • the solid support further comprises a plurality of nucleic acids that hybridize to a plurality of endogenous reference genes selected from the group consisting of USP7, MAPRE2, CSNK1G2, SAFB2, PRKAR2A, PI4KB, CRTC1, HADHA, MAP1LC3B, KAT5, CDC2L1///CDC2L2, GTSE1, TCF25, CHP, LRRC40, hCG — 2003956///LYPLA2///LYPLA2P1, DAXX, UBE2NL, EIF1, KCMF1, PRKRIP1, CHMP4A, TMEM184C, TINF2, PODNL1, FBXO42, LOC441258, RRP1, C10orf104, ZDHHC5, C9orf23, LRRC45, NACC1, LOC100133445///LOC115110, PEX16.
  • a plurality of endogenous reference genes selected from the group consisting of USP7, MAPRE2,
  • the solid support further comprises a plurality of nucleic acids that hybridize to a plurality of ischemic stroke-associated biomarkers selected from the group consisting of FAT3, GADL1, CXADR, RNF141, CLEC4E, TIMP2, ANKRD28, TIMM8A, PTPRD, CCRL1, FCRL4, DLX6, GABRB2, GYPA, PHTF1, CKLF, CKLF, RRAGD, CLEC4E, CKLF, FGD4, CPEB2, LOC100290882, UBXN2B, ENTPD1, BST1, LTB4R, F5, IFRD1, KIAA0319, CHMP1B, MCTP1, VNN3, AMN1, LAMP2, FCHO2, ZNF608, REM2, QKI, RBM25, FAR2, ST3GAL6, HNRNPH2, GAB1, UBR5, VAPA, MCTP1, SH3GL3, PGM5, CCDC144C///LOC100134159, LECT2, S
  • the solid support further comprises a plurality of nucleic acids that hybridize to a plurality of cardioembolic stroke-associated biomarkers selected from the group consisting of IRF6, ZNF254, GRM5, EXT2, AP3S2, PIK3C2B, ARHGEF5, COL13A1, PTPN20A///PTPN20B, LHFP, BANK1, HLA-DOA, EBF1, TMEM19, LHFP, FCRL1, OOEP, LRRC37A3, LOC284751, CD46, ENPP2, C19orf28, TSKS, CHURC1, ADAMTSL4, FLJ40125, CLEC18A, ARHGEF12, C16orf68, TFDP1 and GSTK1.
  • a plurality of cardioembolic stroke-associated biomarkers selected from the group consisting of IRF6, ZNF254, GRM5, EXT2, AP3S2, PIK3C2B, ARHGEF5, COL13A1, PTPN20A/
  • the solid support further comprises a plurality of nucleic acids that hybridize to a plurality of carotid stenosis-associated biomarkers selected from the group consisting of NT5E, CLASP2, GRM5, PROCR, ARHGEF5, AKR1C3, COL13A1, LHFP, RNF7, CYTH3, EBF1, RANBP10, PRSS35, C12orf42, LOC100127980, FLJ31945, LOC284751, LOC100271832, MTBP, ICAM4, SHOX2, DOPEY2, CMBL, LOC146880, SLC20A1, SLC6A19, ARHGEF12, C16orf68, GIPC2 and LOC100144603.
  • a plurality of carotid stenosis-associated biomarkers selected from the group consisting of NT5E, CLASP2, GRM5, PROCR, ARHGEF5, AKR1C3, COL13A1, LHFP, RNF7, CYTH
  • the solid support further comprises a plurality of nucleic acids that hybridize to a plurality of atrial fibrillation-associated biomarkers selected from the group consisting of SMC1A, SNORA68, GRLF1, SDC4, HIPK2, LOC100129034, CMTM1, TTC7A, LRRC43, MIF///SLC2A11, PER3, PPIE, COL13A1, DUSP16, LOC100129034, BRUNOL6, GPR176, C6orf164 and MAP3K71P1.
  • the solid support further comprises a plurality of nucleic acids that hybridize to a plurality of transient ischemic attack-associated biomarkers selected from the group consisting of GABRB2, ELAVL3, COL1A1, SHOX2, GABRB2, TWIST1, DPPA4, DKFZP434P211, WIT1, SOX9, DLX6, ANXA3, EPHA3, SOX11, SLC26A8, CCRL1, FREM2, STOX2, ZNF479, LOC338862, ASTN2, FOLH1, SNX31, KREMEN1, ZNF479, ALS2CR11, FIGN, RORB, LOC732096, GYPA, ALPL, LHX2, GALNT5, SRD5A2L2, GALNT14, OVOL2, BMPR1B, UNC5B, ODZ2, ALPL, RASAL2, SHOX, C19orf59, ZNF114, SRGAP1, ELAVL2, NCRNA000
  • the solid support is a microarray.
  • the microarray has 1000 or fewer hybridizing nucleic acids, for example, 900, 800, 700, 600, 500 or fewer hybridizing nucleic acids.
  • the microarray does not comprise nucleic acids that hybridize to genes whose expression is not correlative of or associated with ischemia.
  • 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), hemorrhagic ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage.
  • 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 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.
  • 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.
  • TIA transient ischemic attack
  • mini-stroke 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.
  • a TIA resolves within 24 hours, but most TIA symptoms resolve within 1 hour. 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 minutes and with resolution of most symptoms within 60 minutes.
  • Reference expression profile refers to the pattern of expression of a set of genes (e.g., a plurality of the genes set forth in Tables 3 and 4) differentially expressed (i.e., overexpressed or underexpressed) in ischemia relative to a control (e.g., the expression level in an individual free of an ischemic event or the expression level of a stably expressed endogenous reference biomarker).
  • genes 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 overexpressed in ischemia and a gene 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 underexpressed in ischemia.
  • 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 or more (e.g., genes).
  • a plurality refers to concurrent or sequential determination of 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 or 100, or more, genes.
  • “plurality” refers to all genes listed in one or more tables, e.g., all genes listed in Tables 3 and 4.
  • 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 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).
  • 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.
  • 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).
  • 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).
  • nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • 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)).
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • 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 T m 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 T m , 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.
  • destabilizing agents such as formamide.
  • 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.
  • isolated refers 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • 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.
  • 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, ⁇ -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.
  • 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.
  • 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
  • TGG which is ordinarily the only codon for tryptophan
  • 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.
  • 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 an ischemia-associated gene (e.g., a gene set forth in Tables 3 and 4), 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.
  • an ischemia-associated gene e.g., a gene set forth in Tables 3 and 4
  • 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, or over the full length of the sequence.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • 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.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of nucleic acids and proteins to ischemia-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.
  • 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 (on the internet at 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).
  • 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 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.
  • P(N) the smallest sum probability
  • 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.
  • 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.
  • 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).
  • host cell is meant a cell that contains an expression vector and supports the replication or expression of the expression vector.
  • Host cells may be, for example, prokaryotic cells such as E. coli or eukaryotic cells such as yeast cells or mammalian cells such as CHO cells.
  • Inhibitors “Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity, e.g., ligands, agonists, antagonists, and their homologs and mimetics.
  • modulator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., inhibit expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., induce or activate the expression of a polypeptide or polynucleotide of the invention or bind to, stimulate, increase, open, activate, facilitate, enhance activation or enzymatic activity, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists.
  • Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Assays to identify inhibitors and activators include, e.g., applying putative modulator compounds to cells, in the presence or absence of a polypeptide or polynucleotide of the invention and then determining the functional effects on a polypeptide or polynucleotide of the invention activity.
  • Samples or assays comprising a polypeptide or polynucleotide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is about 80%, optionally 50% or 25-1%.
  • Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi, oligonucleotide, etc.
  • the test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity.
  • Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • a fusion partner e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties.
  • new chemical entities with useful properties are generated by identifying a test compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • HTS high throughput screening
  • a “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 Daltons and less than about 2500 Daltons, preferably less than about 2000 Daltons, preferably between about 100 to about 1000 Daltons, more preferably between about 200 to about 500 Daltons.
  • FIGS. 1A and 1B illustrate how gene expression analysis can be used to distinguish lacunar from non-lacunar stroke.
  • 1 A provides an illustrative cluster plot of 41 probesets corresponding to 40 genes that discriminate lacunar from non-lacunar stroke. Genes are shown on the y-axis and strokes of lacunar, and non-lacunar (arterial and cardioembolic) etiologies are shown on the x-axis. Up regulated genes are shown in red, and down regulated genes in blue. 1 B illustrates the fold change of the 41 probesets. There is a group of genes that are down-regulated in lacunar stroke and up regulated in non-lacunar stroke. Similarly, there is a group of genes that are up-regulated in lacunar stroke and down regulated in non-lacunar stroke. These genes can be used to discriminate lacunar from non-lacunar stroke.
  • FIG. 2 illustrates box and whisker plots of the gene expression values for the 41 probesets that distinguished lacunar stroke from non-lacunar stroke. Lacunar stroke is shown in orange (upper plot), and non-lacunar stroke in green (lower plot). Each probeset demonstrates significant difference in gene expression between groups. However, no single probeset is able to completely separate every single patient with lacunar stroke from every patient with non-lacunar stroke. By combining information from multiple genes in the profile, the separation of lacunar from non-lacunar stroke is achieved for nearly all patients studied.
  • FIG. 3 illustrates a receiver operating characteristics (ROC) curve of the 41 probesets (40 genes) showing the sensitivity and specificity at various instance probabilities for the prediction of lacunar versus non-lacunar stroke.
  • ROC receiver operating characteristics
  • FIGS. 4A and 4B illustrate a probability plot of the predicted diagnosis of lacunar and non-lacunar stroke based on 10-fold cross-validation analysis using the linear discriminant analysis model for the 41 probesets (40 genes).
  • 4 A illustrates the predicted probability of lacunar and non-lacunar stroke in the 30 patients diagnosed clinically as lacunar stroke. Eight subjects were predicted to have a gene expression profile similar to those of non-lacunar stroke, and 22 were predicted to be lacunar stroke.
  • 4 B illustrates the predicted probability of non-lacunar and lacunar stroke in the 86 patients with non-lacunar stroke. Eighty of the 86 were correctly predicted to be non-lacunar stroke.
  • Small deep infarcts including lacunar stroke account for greater than one quarter of all ischemic strokes and are associated with increased risk of cardiovascular disease and dementia.
  • lipohyalinosis of small penetrating arteries is the most common cause, emboli of arterial or cardiac origin are also causes. Determining which small deep infarcts are caused by lacunar disease, arterial emboli, or cardiac emboli is challenging, but nevertheless important to deliver optimal stroke prevention therapy.
  • Lacunar stroke was defined as a lacunar syndrome associated with infarction ⁇ 15 mm of the striatum, internal capsule, corona radiata, thalamus or pons.
  • the present invention is based, in part, on the discovery that gene expression profiles can distinguish lacunar from non-lacunar strokes. Small deep infarcts of unclear cause were frequently predicted to be of non-lacunar etiology; subsequent work-up and analysis can be performed to identify potential cardioembolic and/or arterial causes. Gene expression profiling may also be used to determine the clinical and treatment implications of small deep infarcts predicted to be of non-lacunar etiology.
  • the present invention is based, in part, on the discovery that RNA expression profiling can be used to differentiate stroke of lacunar etiology from non-lacunar stroke.
  • the invention provides a list of 41 probesets (corresponding to 40 genes) that have greater than 90% sensitivity and specificity to distinguish lacunar stroke from non-lacunar strokes.
  • the genes identified herein to be associated with the occurrence of and/or risk of experiencing lacunar stroke find use to diagnose lacunar stroke based upon an RNA expression profile.
  • the use of the presently identified gene allow for the use of a blood test for the rapid diagnosis of a lacunar cause of stroke.
  • the level of expression of genes associated with the occurrence or risk of lacunar stroke can be measured in the blood of patients with an ischemic stroke.
  • the expression of these genes can be assessed using any applicable method in the art, including, e.g., RT-PCR, microarrays or other technology.
  • the expression of these target genes can be normalized to internal control genes, which are known in the art.
  • a panel of control genes that are specific for ischemic stroke have been developed and are quite reliable.
  • the endogenous control genes have fairly constant expression over many age groups, different diseases, and both genders.
  • RNA expression levels of the target genes i.e., lacunar stroke-associated genes
  • the target gene expression is normalized to the control genes.
  • the expression levels of the normalized target genes can then be applied to a linear discriminant analysis model to predict whether the blood sample is from a patient who has experience or is at risk of experiencing lacunar stroke and the probablility that this is the case. Determining the expression levels of the presently identified lacunar stroke-associated genes, the sensitivity and specificity for prediction is greater than 90% for lacunar versus non-lacunar stroke.
  • a blood test for the diagnosis of stroke is useful in several situations.
  • the gene expression panel can be used to predict whether lacunar stroke is the cause of stroke in patients with small deep infarcts of the brain. About 25% of all stroke patients have small deep infarcts. ‘These small deep infarcts can be caused by lacunar small vessel disease, but also by atherosclerotic disease or larger parent arteries and embolism form a cardiac source. An embolic atherosclerotic cause of stroke warrants a different investigative strategy and treatment than a lacunar small vessel cause of stroke.
  • lacunar stroke-associated genes Currently, patients presenting with a small deep infarct are mostly labeled lacunar, and treated with anti-platelet agents. A diagnosis based on the expression levels of the presently identified lacunar stroke-associated genes would identify the lacunar and non-lacunar strokes, and thus guide appropriate treatment for stroke patients. The presently identified lacunar stroke-associated genes further find use for diagnosing patients who have experienced or are at risk of experiencing Transient Ischemic Attacks. In these patients, the cause is often unknown. Thus, a blood test predicting the cause of the TIA could help prevent or ameliorate strokes in these patients.
  • lacunar stroke presently requires a neurologist to take a history, perform an examination and then confirm using X-ray computed tomography (CT) or magnetic resonance imaging (MRI) brain scanning, in addition to vascular imaging of the head and neck with doppler or magnetic resonance angiography (MRA) or CT angiography (CTA); an electrocardiogram (EKG or ECG), cardiac ultrasound and cardiac monitoring; and a series of blood tests.
  • CT computed tomography
  • MRA magnetic resonance angiography
  • CTA CT angiography
  • EKG or ECG electrocardiogram
  • ischemic stroke Individuals who will benefit from the present methods may be exhibiting symptoms of ischemic stroke, and in particular, a small deep infarct (SDI).
  • SDI small deep infarct
  • the subject has experienced an ischemic event.
  • the subject may have suffered or be currently experiencing a small deep infarct, a transient ischemic attack (TIA), an ischemic stroke, a myocardial infarction, peripheral vascular disease, or venous thromboembolism.
  • the subject may have or have been diagnosed with cerebral vascular disease.
  • the subject may be suspected of having experienced an ischemic event, and in particular, a small deep infarct (SDI).
  • Brain imaging on the patient may indicate microhemorrhage and/or blood-brain permeability.
  • the levels of expression of the panel of biomarkers is determined within 3 hours of a suspected ischemic event. In some embodiments, the levels of expression of the panel of biomarkers is determined at 3 or more hours after a suspected ischemic event. In some embodiments, the levels of expression of the panel of biomarkers is determined within 6, 12, 18, 24, 36, 48 or 72 hours of a suspected ischemic event.
  • the subject is asymptomatic, but may have a risk or predisposition to experiencing ischemic stroke, e.g., based on genetics, familial history, a related disease condition, environment or lifestyle.
  • the patient has one or more vascular risk factors, e.g., hypertension, diabetes mellitus, hyperlipidemia, or tobacco smoking.
  • the subject is non-Caucasian, for example, Asian, African-American or Latino or of Asian, African-American or Latino descent.
  • Ataxia 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 abnormatities 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.
  • Biomarkers useful for the prediction, diagnosis or confirmation of the occurrence of lacunar stroke, or for distinguishing lacunar stroke from non-lacunar stroke are listed in Tables 3 and 4.
  • Determination of the expression levels of a plurality of the biomarkers of Tables 3 and/or 4 can be performed for the prediction, diagnosis or confirmation of the occurrence of lacunar stroke in conjunction with other biomarkers known in the art for the prediction, diagnosis or confirmation of the occurrence of ischemic stroke, SDI and/or lacunar stroke, in conjunction with other methods known in the art for the diagnosis of ischemic stroke, SDI and/or lacunar 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.
  • Determination of the expression levels of a plurality of the biomarkers of Tables 3 and/or 4 can be performed for the prediction, diagnosis or confirmation of the occurrence of stroke can also be performed independently, e.g., to diagnose that a lacunar stroke has occurred, to distinguish lacunar stroke from non-lacunar stroke or non-lacunar SDI, or to determine the risk that a patient may suffer a lacunar stroke.
  • the expression levels of at least about 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 or more biomarkers from Table 3 or Table 4 are determined. In some embodiments, the expression levels of a plurality of biomarkers in Table 3 or Table 4 are determined. In some embodiments, the expression levels of all listed biomarkers in Table 3 or Table 4 are determined.
  • the level of expression of biomarkers indicative of the occurrence of lacunar stroke is determined within 72 hours, for example, within 60, 48, 36, 24, 12, 6 or 3 hours of a suspected ischemic event.
  • a decreased expression level of one or more lacunar stroke-associated biomarkers of Table 3 selected from the group consisting of AKAP9, ALS2CR11, BNC2, BZRAP1, C18orf49, CALM1, CCDC114, CCDC78, CCL2, CCL3, CCL3L1, CCL3L3, CCL4, CHST2, CSF1, ERBB2, FAM179A, GBP4, GBR56, GRAMD3, GRHL2, GRK4, HLA-DRB4, ITIH4, KIAA1618, LAG3, LAIR2, LGR6, LOC100132181, LOC147646, LOC150622, LOC161527, OASL, PLEKHF1, PRKD2, PROCR, PRSS23, RASEF, RGNEF, RUNX3, SCAND2, SESN2, SLAMF7, SPON2, STAT1, SYNGR1, TRX21, TGFBR3, TMEM67, TSEN54, TTC12, TUBE1, UBA7, UTS2, and ZNF
  • the level of expression of biomarkers indicative of the occurrence of lacunar stroke is determined within 72 hours, for example, within 60, 48, 36, 24, 12, 6 or 3 hours of a suspected ischemic event.
  • a decreased expression level of one or more lacunar stroke-associated biomarkers of Table 4 selected from the group consisting of STK4, LRRC8B, PDXDC1, LOC254128, IL8, GTF2H2, UGCG, MPZL3, VAPA, STX7, FAM70B, QKI, CHML, FLJ13773, HLA-DQA1 is correlative with or indicates that the patient suffers from or is at risk of developing lacunar stroke.
  • an increased expression level of one or more lacunar stroke-associated biomarkers of Table 4 selected from the group consisting of STK4, LRRC8B, PDXDC1, LOC254128, IL8, GTF2H2, UGCG, MPZL3, VAPA, STX7, FAM70B, QKI, CHML, FLJ13773, HLA-DQA1 is correlative with or indicates that the patient suffers from or is at risk of developing non-lacunar stroke.
  • 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.
  • the control level of expression can be from a population of individuals known to not have or be at risk for an ischemic event such as lacunar stroke or can be determined with reference to a panel of stably expressed reference biomarkers.
  • 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 event such as lacunar stroke versus known to have or be at risk for lacunar stroke).
  • the biological sample may be tested for expression levels of biomarkers useful for distinguishing lacunar stroke from non-lacunar stroke, as well as for expression levels of biomarkers useful for the determination of the cause of ischemic stroke, particularly non-lacunar stroke. Measuring the expression levels of biomarkers to diagnose the cause of non-lacunar stroke can be performed concurrently with (i.e., in parallel) or sequentially to measuring the expression levels of biomarkers to distinguish the cause of stroke as lacunar or non-lacunar.
  • Biomarkers useful for the determination and diagnosis of the cause of stroke are described, e.g., in co-owned and co-pending Application Nos. 61/364,334 and 61/364,449, the disclosures of both of which are hereby incorporated herein by reference in their entirety for all purposes.
  • the expression levels of a plurality of biomarkers can be measured to determine whether a suspected or predicted ischemic event is cardioembolic or atherosclerotic.
  • 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 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 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.
  • Overexpression or underexpression of a plurality of ischemic-stroke-associated biomarkers that is at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold or 3.5-fold, or more, in comparison to the expression levels of a plurality of stably expressed endogenous reference biomarkers, e.g., those listed in Table 1, is correlative with or indicates that the subject has experienced or is at risk of experiencing ischemic stroke.
  • Overexpression or underexpression of a plurality of biomarkers that is at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold or 3.5-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 or ischemic event is correlative with or indicates that the subject has experienced or is at risk of experiencing ischemic stroke.
  • the expression levels of a plurality of lacunar stroke associated gene 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.
  • 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), ARHGEF5 (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 — 080805/
  • the expression levels of a plurality of lacunar stroke associated gene 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).
  • ischemia e.g., a transient ischemic attack
  • a thromboembolic stroke a.k.a., atherothrombosis, large-artery atherosclerosis, atherosclerosis with stenosis.
  • an increased expression level of one or more ischemic stroke-associated biomarkers selected from the group consisting of NT5E (NM — 002526), CLASP2 (NM — 015097), GRM5 (NM — 000842///NM — 001143831), PROCR(NM — 006404), ARHGEF5 (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////////
  • 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
  • the expression levels of a plurality of lacunar stroke associated gene 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
  • 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.
  • 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.
  • 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 — 080805///
  • the expression levels of a plurality of lacunar stroke associated gene 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).
  • TIA transient ischemic attacks
  • 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.
  • 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///////
  • 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 — —
  • the expression levels of the lacunar stroke-associated biomarkers are compared to a control level of expression.
  • the control level of expression can be the expression level of the same lacunar stroke-associated biomarker in an otherwise healthy individual (e.g., in an individual who has not experienced and/or is not at risk of experiencing a vascular event, e.g., TIA, ischemic stroke or a small deep infarct).
  • the control level of expression is the expression level of a plurality of stably expressed endogenous reference biomarkers, as described herein and/or known in the art.
  • control level of expression is a predetermined threshold level of expression of the same lacunar stroke-associated biomarker, e.g., based on the expression level of the biomarker in a population of otherwise healthy individuals.
  • expression level of the lacunar stroke-associated biomarker in the test subject and the expression level of the lacunar stroke-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.
  • the overexpression or underexpression of a lacunar stroke associated biomarker is determined with reference to the expression of the same lacunar stroke associated biomarker in an otherwise healthy individual.
  • a healthy or normal control individual has not experienced and/or is not at risk of experiencing ischemic stroke, transient ischemic attack or a small deep infarction.
  • the healthy or normal control individual generally has not experienced a vascular event (e.g., TIA, ischemic stroke, myocardial infarction, peripheral vascular disease, or venous thromboembolism) and does not have cerebral vascular disease.
  • 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).
  • the expression levels of the target lacunar stroke-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.
  • the overexpression or underexpression of a lacunar stroke 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 with stroke or TIAs or SDIs as compared to control patients.
  • Target biomarkers are expressed at higher or lower levels in the blood of the stroke or TIA or SDI patients.
  • 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 endogenous reference biomarkers.
  • the normalized expression level of a target biomarker can be used to predict the occurrence or lack thereof of stroke or TIA or SDI, and/or the cause of stroke or TIA or SDI.
  • the expression level of the lacunar stroke-associated biomarker from a patient suspected of having or experiencing lacunar stroke and from a control patient are normalized with respect to the expression levels of a plurality of stably expressed endogenous genes.
  • the expression levels of the normalized expression of the lacunar stroke-associated biomarker can be compared to the expression levels of the normalized expression of the same lacunar stroke-associated biomarker in a control patient.
  • the determined fold change in expression normalized expression of target biomarker in lacunar stroke patient/normalized expression of target biomarker in control patient.
  • the control level of expression is a predetermined threshold level.
  • the threshold level can correspond to the level of expression of the same lacunar stroke-associated biomarker in an otherwise healthy individual or a population of otherwise healthy individuals, optionally normalized to the expression levels of a plurality of endogenous reference biomarkers. After expression levels and normalized expression levels of the lacunar stroke-associated biomarkers are determined in a representative number of otherwise healthy individuals and individuals predisposed to experiencing SDI or lacunar stroke, normal and lacunar stroke expression levels of the lacunar stroke-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 lacunar stroke or the occurrence of lacunar stroke.
  • the predetermined threshold level of expression is with respect to a population of normal control patients, then overexpression or underexpression of the lacunar stroke-associated biomarker (usually normalized) in the stroke patient by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold or 3.5-fold, or more, in comparison to the threshold level is correlative with or indicates that the lacunar stroke patient has experienced or is at risk of experiencing lacunar stroke.
  • an expression level in the patient suspected of experiencing lacunar stroke that is approximately equal to the threshold level (or overexpressed or underexpressed greater than the threshold level of expression), is correlative with or indicates that the lacunar stroke or SDI patient has experienced or is at risk of experiencing lacunar stroke.
  • endogenous reference biomarkers preferably, Exemplary endogenous reference biomarkers that find use are listed in Table 1, below. Further suitable endogenous reference biomarkers are published, e.g., in Stamova, et al., BMC Medical Genomics (2009) 2:49.
  • the expression levels of a plurality of 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, endogenous reference biomarkers, e.g., as listed in Table 1 or known in the art, are determined as a control.
  • the expression levels of the endogenous reference biomarkers GAPDH, ACTB, B2M, HMBS and PPIB are determined as a control.
  • Biomarkers indicative of lacunar stroke have levels of expression that are at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold or 3.5-fold, or more, in comparison to the expression levels of a plurality of stably expressed endogenous reference biomarkers, e.g., the geometric average expression level of the evaluated endogenous reference biomarkers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or more biomarkers listed in Table 1.
  • the geometric average expression level of the evaluated endogenous reference biomarkers e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or more biomark
  • 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.
  • 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, Sambrook, supra and Ausubel, supra) and may be used to detect the expression of the genes set forth in Tables 3 and 4. 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.
  • RFLP restriction fragment length polymorphism
  • All forms of RNA can be detected, including, e.g., message RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA).
  • mRNA message RNA
  • miRNA microRNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • nucleic acid hybridization format is not critical.
  • a variety of nucleic acid hybridization formats are known to those skilled in the art.
  • 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.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • 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., Tijssen, “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.
  • 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).
  • 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 3 H, 125 I, 35 S, 14 C, or 32 P-labeled probes or the like.
  • 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.
  • 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, NY (1997); and in Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).
  • 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.
  • the amount of RNA is measured by quantifying the amount of label fixed to the solid support by binding of the detection reagent.
  • 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.
  • 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.
  • 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 consists of a reproducible pattern of a plurality of nucleic acids (e.g., a plurality of nucleic acids that hybridize to a plurality of the genes set forth in Tables 3 and 4) attached to a solid support.
  • the array contains a plurality of nucleic acids that hybridize to a plurality of the genes listed in Table 3.
  • the array contains a plurality of nucleic acids that hybridize to a plurality of the genes listed in Table 4.
  • the array further contains a plurality of nucleic acids that hybridize to a plurality of genes useful for diagnosing ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation or transient ischemic attacks, as described herein or known in the art.
  • 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 ischemia (e.g., stroke or SDI (lacunar or non-lacunar) or transient ischemic attacks).
  • 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).
  • VLSIPSTM very large scale immobilized polymer arrays
  • Affymetrix, Inc. 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.
  • Microfluidics systems for use in detecting levels of expression of a plurality of nucleic acids are available, e.g., from NanoString Technologies, on the internet at nanostring.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).
  • a labeled detection moiety that binds specifically to duplex nucleic acids
  • a labeled detection moiety that binds specifically to duplex nucleic acids
  • 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.
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a dissociation constant (K D ) of at least about 0.1 ⁇ M, preferably at least about 0.01 ⁇ M or better, and most typically and preferably, 0.001 ⁇ M or better.
  • K D dissociation constant
  • 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.
  • 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.
  • a nucleic acid amplification system that multiplies the target nucleic acid being detected.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • 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.
  • 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).
  • 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).
  • 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.
  • quantitative RT-PCR is used to detect the expression of a plurality of the genes set forth in Tables 3 and 4. In one embodiment, quantitative RT-PCR is used to detect a plurality of the genes listed in Table 3. In one embodiment, quantitative RT-PCR is used to detect a plurality of the genes listed in Table 4. In one embodiment, quantitative RT-PCR is used to further detect a plurality of the genes useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation and/or transient ischemic attacks, as described herein and known in the art.
  • the invention provides 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 Tables 3 and 4.
  • the invention provides 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 Table 3.
  • the invention provides 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 Table 4.
  • 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 and/or transient ischemic attacks, as described herein and known in the art.
  • 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.
  • 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 Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994-2008, Wiley Interscience)).
  • 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.
  • kb kilobases
  • bp base pairs
  • 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).
  • 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.
  • the invention also provides expression reference profiles useful for the diagnosis of lacunar stroke or for distinguishing lacunar stroke from non-lacunar stroke.
  • the gene expression reference profiles comprise information correlating the expression levels of a plurality of lacunar stroke-associated genes (i.e., a plurality of the genes set forth in Tables 3 and 4) to lacunar stroke (versus non-lacunar stroke).
  • the lacunar stroke reference profile correlates the expression levels of a plurality of the genes listed in Table 3 to the occurrence or risk of lacunar stroke.
  • the lacunar stroke reference profile correlates the expression levels of a plurality of the genes listed in Table 4 to the occurrence or risk of lacunar stroke.
  • the lacunar stroke reference profile correlates the expression levels of a plurality of the genes listed in Table 3 to the occurrence or risk of non-lacunar stroke (e.g., cardioembolic stroke, carotid stenosis, atrial fibrillation, transient ischemic attacks, or other causes). In one embodiment, the lacunar stroke reference profile correlates the expression levels of a plurality of the genes listed in Table 4 to the occurrence or risk of non-lacunar stroke (e.g., cardioembolic stroke, carotid stenosis, atrial fibrillation, transient ischemic attacks, or other causes).
  • the profiles can conveniently be used to diagnose, monitor and prognose the cause of an ischemic event.
  • the lacunar stroke reference profile correlates the expression levels of a plurality of the genes selected from Table 3.
  • the lacunar stroke reference profile correlates the expression levels of a plurality of the genes selected from Table 4.
  • One embodiment of the invention further provides an ischemia reference profile for subjects who have experienced or are at risk for experiencing stroke, regardless of cause.
  • the ischemia reference profile correlates the expression levels of a plurality of ischemic stroke-associated genes.
  • One embodiment of the invention further provides an ischemia reference profile for subjects who have experienced or are at risk for experiencing cardioembolic stroke. Accordingly, the ischemia reference profile correlates the expression levels of a plurality of the genes correlative for or associated with cardioembolic stroke.
  • One embodiment of the invention further provides an ischemia reference profile for subjects who have experienced or are at risk for experiencing carotid stenosis and atherosclerotic stroke. Accordingly, the ischemia reference profile correlates the expression levels of a plurality of the genes correlative for or associated with carotid stenosis and atherosclerotic stroke.
  • One embodiment of the invention further provides an ischemia reference profile for subjects who have experienced or are at risk for experiencing atrial fibrillation. Accordingly, the ischemia reference profile correlates the expression levels of a plurality of the genes correlative for or associated with atrial fibrillation.
  • an expression profile exhibiting an increase in expression of a plurality of the following genes: SMC1A, SNORA68, GRLF1, SDC4, HIPK2, LOC100129034, CMTM1 and TTC7A when compared to the control level, and/or a decrease in expression of a plurality of the following genes: LRRC43, MIF///SLC2A11, PER3, PPIE, COL13A1, DUSP16, LOC100129034, BRUNOL6, GPR176, C6orf164 and MAP3K71P1 when compared to the control level is a reference profile for a subject who has experienced or is at risk for atrial fibrillation.
  • One embodiment of the invention further provides an ischemia reference profile for subjects who have experienced or are at risk for experiencing transient ischemic attacks. Accordingly, the ischemia reference profile correlates the expression levels of a plurality of the genes correlative for or associated with transient ischemic attacks.
  • the reference profiles can be entered into a database, e.g., a relational database comprising data fitted into predefined categories.
  • a database e.g., a relational database comprising data fitted into predefined categories.
  • Each table, or relation contains one or more data categories in columns.
  • Each row contains a unique instance of data for the categories defined by the columns.
  • a typical database for the invention would include a table that describes a sample with columns for age, gender, reproductive status, expression profile and so forth. Another table would describe a disease: symptoms, level, sample identification, expression profile and so forth.
  • the invention matches the experimental sample to a database of reference samples.
  • the database is assembled with a plurality of different samples to be used as reference samples. An individual reference sample in one embodiment will be obtained from a patient during a visit to a medical professional.
  • Information about the physiological, disease and/or pharmacological status of the sample will also be obtained through any method available. This may include, but is not limited to, expression profile analysis, clinical analysis, medical history and/or patient interview. For example, the patient could be interviewed to determine age, sex, ethnic origin, symptoms or past diagnosis of disease, and the identity of any therapies the patient is currently undergoing. A plurality of these reference samples will be taken. A single individual may contribute a single reference sample or more than one sample over time.
  • confidence levels in predictions based on comparison to a database increase as the number of reference samples in the database increases.
  • the database is organized into groups of reference samples. Each reference sample contains information about physiological, pharmacological and/or disease status.
  • the database is a relational database with data organized in three data tables, one where the samples are grouped primarily by physiological status, one where the samples are grouped primarily by disease status and one where the samples are grouped primarily by pharmacological status. Within each table the samples can be further grouped according to the two remaining categories. For example the physiological status table could be further categorized according to disease and pharmacological status.
  • the present invention may be embodied as a method, data processing system or program products. Accordingly, the present invention may take the form of data analysis systems, methods, analysis software, etc.
  • Software written according to the present invention is to be stored in some form of computer readable medium, such as memory, hard-drive, DVD ROM or CD ROM, or transmitted over a network, and executed by a processor.
  • the present invention also provides a computer system for analyzing physiological states, levels of disease states and/or therapeutic efficacy.
  • the computer system comprises a processor, and memory coupled to said processor which encodes one or more programs.
  • the programs encoded in memory cause the processor to perform the steps of the above methods wherein the expression profiles and information about physiological, pharmacological and disease states are received by the computer system as input.
  • Computer systems may be used to execute the software of an embodiment of the invention (see, e.g., U.S. Pat. No. 5,733,729).
  • the methods further provide for the step of prescribing, providing or administering a regime for the prophylaxis or treatment of ischemic stroke or SDI.
  • a patient can rapidly receive treatment that is tailored to and appropriate for the type of stroke that has been experienced, or that the patient is at risk of experiencing.
  • 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.,
  • the patient may be administered tissue plasminogen activator within three hours of an ischemic event if the patient is without contraindications (i.e. a bleeding diathesis such as recent major surgery or cancer with brain metastases).
  • High dose aspirin may be given within 48 hours of an ischemic event.
  • medical regimens may be aimed towards correcting the underlying risk factors for lacunar infarcts such as hypertension, diabetes mellitus and cigarette smoking.
  • ischemic stroke-associated biomarkers indicate the occurrence or risk of ischemic stroke
  • a positive diagnosis of ischemic stroke can be supported or confirmed using methods known in the art.
  • the patient can be subject to MRI imaging of brain and vessels, additional blood tests, EKG, and/or echocardiogram.
  • the patient can be prescribed or administered a regime of an anticoagulant.
  • anticoagulants include aspirin, heparin, warfarin, and dabigatran.
  • 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).
  • Plavix anti-platelet drug clopidogrel
  • Some studies indicate that aspirin is most effective in combination with another anti-platelet drug.
  • 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.
  • 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.
  • the patient can be prescribed a regime of an anti-coagulant (to prevent stroke) and/or a pharmacological agent to achieve rate control.
  • anticoagulants include aspirin, heparin, warfarin, and dabigatran.
  • 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).
  • 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.
  • the medication selected will depend on the location, cause, severity and type of TIA, if TIA has occurred.
  • 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).
  • the patient is prescribed a combination of low-dose aspirin and the anti-platelet drug dipyridamole (Aggrenox), to reduce blood clotting.
  • Ticlopidine is another anti-platelet medication that finds use to prevent or reduce the risk of stroke in patients who have experienced TIA.
  • the patient may be prescribed a regime of an anticoagulant.
  • 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.
  • the patient may require or benefit from carotid angioplasty, or stenting.
  • the present methods for determining whether a patient has experienced or has a predisposition to experience lacunar stroke can be confirmed, complemented by, and/or used in conjunction with diagnostic tests known in the art for diagnosing lacunar stroke.
  • the present methods can be performed in conjunction with additional diagnostic based on imaging or ultrasound techniques.
  • the present methods are performed in conjunction with one or more diagnostic tests selected from the group consisting of X-ray computed tomography (CT), magnetic resonance imaging (MRI) brain scanning, vascular imaging of the head and neck with doppler or magnetic resonance angiography (MRA), CT angiography (CTA), electrocardiogram (e.g., EKG or ECG), cardiac ultrasound and cardiac monitoring.
  • CT computed tomography
  • MRA magnetic resonance angiography
  • CTA CT angiography
  • electrocardiogram e.g., EKG or ECG
  • cardiac ultrasound and cardiac monitoring e.g., EKG or ECG
  • the patient is subjected to cardiac monitoring for at least 2 days, e.g., for 2-30 days or for 7-21 days, e.g., for 2, 5, 7, 10, 12, 14, 18, 20, 21, 25, 28, 30, or more days, as appropriate.
  • An infarction located in a subcortical region of the brain is associated with or correlated with a diagnosis of lacunar stroke.
  • An infarction located in a cortical region of the brain e.g., in regions of the penetrating arteries, e.g., basal ganglia, thalamus, internal capsule, corona radiata and/or pons, is associated with or correlated with a diagnosis of non-lacunar stroke.
  • the size of the infarction is determined.
  • 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 genes set forth in Tables 3 and 4, and optionally Table 1.
  • 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 genes set forth in Tables 3 and 4, and optionally Table 1.
  • the solid supports are configured to exclude genes not associated with or useful to the diagnosis, prediction or confirmation of a lacunar stroke, or for stroke generally.
  • genes that are overexpressed or underexpressed less than 1.2-fold in subjects with lacunar stroke in comparison to a control level of expression can be excluded from the present solid supports.
  • genes that are overexpressed or underexpressed less than 1.2-fold in subjects with ischemic stroke, including lacunar stroke, cardioembolic stroke, atherothrombotic stroke, TIA, and stroke subsequent to atrial fibrillation, in comparison to a control level of expression can be excluded from the present solid supports.
  • the solid support may optionally further comprise a plurality of nucleic acid probes that hybridize to a plurality (e.g., two or more, or all) of the genes useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, and/or atrial fibrillation, as described herein.
  • the solid support comprises 1000 or fewer (e.g., 900, 800, 700, 600, 500 or fewer) nucleic acid probes that hybridize to a plurality of ischemia-associated genes, as described herein.
  • the solid support may be a component in a kit.
  • kits for diagnosing ischemia or a predisposition for developing ischemia are provided.
  • the invention provides kits that include one or more reaction vessels that have aliquots of some or all of the reaction components of the invention 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 that hybridize to a plurality (e.g., two or more, or all) of the genes set forth in Tables 3 and 4.
  • the kits comprise a plurality of nucleic acid probes that hybridize to a plurality of the genes set forth in Table 3.
  • kits comprise a plurality of nucleic acid probes that hybridize to a plurality of the genes set forth in Table 4. In one embodiment, the kits further comprise a plurality of nucleic acid probes that hybridize to a plurality of the genes set useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation, and/or transient ischemic attacks (TIA), as described herein.
  • the probes may be immobilized on an array as described herein.
  • 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 genes set forth in Tables 3 and 4.
  • 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 genes set forth in Table 3.
  • 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 genes set forth in Table 4.
  • 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 genes useful for the diagnosis of ischemic stroke, cardioembolic stroke, carotid stenosis, atrial fibrillation, and/or transient ischemic attacks (TIA), as described herein.
  • the kits can also include written instructions for the use of the kit.
  • kits comprise a plurality of antibodies that bind to a plurality of the biomarkers set forth in Tables 3 and 4.
  • 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, and/or transient ischemic attacks (TIA), as described herein.
  • the antibodies may or may not be immobilized on a solid support, e.g., an ELISA plate.
  • Lacunar stroke was defined by clinical symptoms consistent with a lacunar syndrome and evidence of restricted diffusion on MRI with a largest diameter ⁇ 15 mm occurring in the basal ganglia, thalamus, internal capsule, corona radiata or pons. Patients with lacunar stroke did not have evidence of embolic source despite investigation, including no evidence of intracranial or extracranial stenosis >50% or a potential moderate to high risk cardioembolic source. Lacunar strokes with incomplete investigations were not included for study.
  • SDI of unclear etiology were defined as infarction in the basal ganglia, thalamus, internal capsule, corona radiata or brainstem >15 mm in diameter or ⁇ 15 mm with a potential cardioembolic or ipsilateral arterial cause of stroke.
  • Non-lacunar strokes occurred had evidence of infarction on imaging non-lacunar stroke regions, and were of identified cardioembolic or arterial source.
  • Cardioembolic strokes included patients with atrial fibrillation, acute myocardial infarction, valvular heart disease and marked ventricular hypokinesis with hemispheric infarcts. Patients with PFO, atrial myxoma or endocarditis were not included.
  • Arterial strokes were defined as stenosis >50% of an extracranial or intracranial artery referable to the infarct without evidence of other cause of stroke. Differences between groups were analyzed using Fisher's exact test or two-tailed t-test where appropriate.
  • RNA samples were used to collect a venous blood sample within 72 hours of stroke onset (PreAnalytiX, Germany). Samples were stored at ⁇ 80° C. and processed at the same time in the same laboratory to reduce batch effect. Total RNA was isolated according to the manufacturer's protocol (PAXgene blood RNA kit; Pre-AnalytiX). RNA concentration was determined by Nano-Drop (Thermo Fisher) and RNA quality by Agilent 2100 Bioanalyzer. Samples required A260/A280 absorbance ratios of purified RNA ⁇ 2.0 and 28S/18S rRNA ratios ⁇ 1.8.
  • RNA samples were hybridized according to the manufacturer's protocol on Affymetrix Human U133 Plus 2.0 GeneChips (Affymetrix Santa Clara, Calif.). Arrays were washed and processed on a Fluidics Station 450 and scanned on a Genechip Scanner 3000. Samples were randomly assigned to microarray batch stratified by stroke subtype.
  • Microarray data files were pre-processed using robust multichip averaging (RMA), mean-centering standardization and log 2 transformation.
  • RMA multichip averaging
  • Partek Genomics Suite 6.4 Partek Inc., St. Louis, Mo.
  • Nonspecific interquartile range filtering was used to eliminate probesets with low variation ( ⁇ 0.5) across the dataset (Hackstadt and Hess, BMC Bioinformatics (2009) 10:11; Gentleman R., “Bioinformatics and computational biology solutions using R and Bioconductor,” in Statistics for biology and health . New York: Springer Science+Business Media; 2005. p. xix, 473 pp.).
  • 21526 passed this filtering step and were retained for further analysis.
  • Probesets were further selected by the differences in gene expression between phenotypic classes of interest using Analysis of Covariance (ANCOVA), adjusted for age and gender with a false discovery rate ⁇ 0.05 and fold change ⁇
  • ANCOVA Analysis of Covariance
  • Classifier results were obtained using forward selection linear discriminant analysis with a multiple 10-fold cross-validation method comparing lacunar stroke to non-lacunar stroke.
  • data were divided into 10 equal-sized subsamples.
  • Nine of the subsamples were used to predict the cause of stroke in the remaining “left-out” subsample. This procedure was repeated 10 times, each time using a different left-out subsample, so that all patient samples were used to derive and evaluate predictors.
  • the genes used in the classifier were reselected based only on the samples not left out, so that only the training set was used to derive predictors for the left-out subsample.
  • Selected predictors represent genes whose expression is most stable within samples from the same phenotypic class (e.g. lacunar stroke) and whose expression differs the most between samples of a different class.
  • Receiver operating characteristics were derived based on the instance probability of class membership and used to identify the optimal probability threshold to assign class membership to subjects of unknown stroke cause.
  • the full classifier derived from subjects with known stroke subtype was further evaluated using a second validation cohort of subjects of known stroke cause. To predict the stroke subtype in patients with SDI of unclear cause, the full classifier was applied to the gene expression values and class membership assigned based on probability threshold determined from the training set. Logistic regression analyses were performed using Stata 10.1 (College Station, Tex., USA). Variables on univariate analysis with p ⁇ 0.2 were included in multivariate analysis. Results are reported as odds ratios (OR) with 95% confidence intervals.
  • Ingenuity Pathway Analysis (IPA, Ingenuity Systems®, on the internet at ingenuity.com) was used to identify the functional pathways associated with the 90 genes. This was done by testing whether the number of genes in a given pathway was greater than that expected by chance (p ⁇ 0.05 considered significant using a Fisher's exact test).
  • a cluster plot and a plot of fold change for the 41 probesets that distinguish lacunar versus non-lacunar strokes are shown in FIG. 1 .
  • Detailed box plots of the mean centered expression values are shown in FIG. 2 .
  • Receiver Operating Characteristics curve was used to identify 0.7 as the optimal instance probability to discriminate between lacunar and non-lacunar stroke (true positive rate 0.97, false positive rate 0) ( FIG. 3 ).
  • Ten-fold cross-validation analysis was performed to evaluate prediction in the training set.
  • the 41 probesets distinguished lacunar from non-lacunar stroke in 88% of patients (22/30 lacunar strokes; 80/86 non-lacunar strokes) ( FIG. 4 ).
  • NM_207346 0.00299112 1.52859 219587_at TTC12 tetratricopeptide repeat domain 12 NM_017868 0.00822832 1.53053 230891_at TUBE1 Tubulin, epsilon 1 NM_016262 0.00822258 1.57063 223279_s_at UACA uveal autoantigen with coiled-coil domains and ankyrin NM_001008224 /// 0.00474228 ⁇ 1.55924 repeats NM_018003 203281_s_at UBA7 ubiquitin-like modifier activating enzyme 7 NM_003335 0.00155438 1.56379 221765_at UGCG UDP-glucose ceramide glucosyltransferase NM_003358 0.00265623 ⁇ 1.61812 224967_at UGCG UDP-glucose ceramide glucosyltransferase NM_0033
  • the model derived from the training cohort was applied to a second validation test cohort of 36 ischemic stroke subjects of known non-lacunar etiology.
  • the 41 probesets were able to correctly classify 35 of the 36 (98%) strokes as non-lacunar.
  • SDI unclear cause
  • 15 were predicted to be of lacunar etiology
  • 17 were predicted to be of non-lacunar etiology.
  • univariate analysis was performed.
  • SDI predicted to be lacunar were less likely to be of Caucasian race/ethnicity (OR 0.18, 0.04-0.86), less likely to have potential arterial source of stroke (OR 0.2, 0.04-0.9) and trended to have fewer potential cardiac source of stroke (OR 0.28, 0.04-1.69) (Table 5).
  • the presence of hypertension and diabetes were not significantly increased in SDI predicted to be lacunar.
  • Multivariate logistic regression was performed to identify independent predictors of lacunar infarction.
  • Independent predictors of SDI being of lacunar etiology were non-Caucasian race (OR 0.06, 0.005-0.60) and the absence arterial disease ipsilateral to the stroke (OR 0.06, 0.006-0.64).
  • Table 6 shows the results of a multivariate stepwise logistic regression of all variables with p ⁇ 0.2 on univariate analysis to identify independent predictors of lacunar stroke in small deep infarcts of unclear cause.
  • Variables included in the model were arterial source, cardiac source, race Caucasian, striatocapsular location, infarct size, microhemorrhage and hyperlipidemia.
  • Functional analysis of the 96 probesets revealed several pathways that were represented greater than expected by chance. The majority of pathways represented alterations in immune cells in the blood of patients with lacunar stroke. The top ten functional and canonical pathways are listed in Table 7, along with the genes expressed in these pathways. Table 7 shows a functional analysis of the 96 probesets (90 genes) that were different between lacunar and non-lacunar stroke. The top functional and canonical pathways that were represented greater than expected by chance (p ⁇ 0.05, Fisher's exact test), along with the genes expressed in the listed pathways. The majority of pathways represent alterations in immune cells in the blood of patients with lacunar stroke that are different from non-lacunar stroke patients.
  • Symptomatic carotid stenosis derives greater benefit from vascular intervention. Thus ascertaining whether the SDI is of lacunar or arterial etiology is of clinical significance. Furthermore, correct classification of stroke cause is important for clinical research of disease mechanism and the development of therapeutics.
  • Vascular risk factor profiles are similar between lacunar and non-lacunar stroke (Jackson, et al. Stroke (2010) 41 (4):624-9; Jackson and Sudlow, Stroke (2005) 36 (4):891-901; Jackson and Sudlow, Brain (2005) 128 (Pt 11):2507-17; and Bejot, et al., Stroke (2008) 39 (7):1945-51).
  • This is consistent with our study which did not identify hypertension or diabetes as being associated with a predicted diagnosis of lacunar infarction.
  • non-Caucasian race/ethnicity was identified as being more common in SDI predicted to be of lacunar etiology.
  • lacunar stroke occurs more frequently in non-Caucasians, including African American, Asian and Latino (Gross, et al., Stroke (1984) 15 (2):249-55; Bamford, et al., Stroke (1987) 18 (3):545-51; Bogousslaysky, et al., Stroke (1988) 19 (9):1083-92; Huang, et al., Stroke (1990) 21 (2):230-5, and Ohira, et al., Stroke (2006) 37 (10):2493-8).
  • non-Caucasian strokes also tend to have more intracranial atherosclerotic disease (Sacco, et al., Stroke (1997) 28 (5):929-35; Sacco, Stroke (1995) 26 (1):14-20; Gorelick, Stroke (1993) 24 (12 Suppl):I16-9; discussion 120-1; Caplan, et al., Stroke (1986) 17 (4):648-55).
  • race is an indicator of intracranial vascular disease not detected by angiography that is associated with lacunar stroke.
  • microhemorrhages may be an important marker of lacunar stroke has previously been reported (Wardlaw, et al., Stroke (2006) 37 (10):2633-6; Fan, et al., J Neurol . (2004) 251 (5):537-41, Schonewille, et al., J Stroke Cerebrovasc Dis . (2005) 14 (4):141-4).
  • small vessel disease markers including microhemorrhage, retinal imaging, blood brain barrier permeability and blood endothelial markers may provide better insight into features characteristic of lacunar stroke.
  • the identified differences in blood reflect immune differences between lacunar and embolic stroke, including differences in immune response to vascular risk factors.
  • the genes identified as differentially expressed in lacunar stroke were over represented in canonical pathways involving innate and adaptive immune cell communication, TREM1 signaling, T-helper cell differentiation and immune cell signaling (Table 7). Over represented functional pathways included growth, activation and recruitment of leukocytes and myeloid cells, endothelial adhesion and angiogenesis. Specific inflammatory and/or genetic factors may predispose to endothelial damage.

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US9803243B2 (en) 2010-07-15 2017-10-31 The Regents Of The University Of California Biomarkers for diagnosis of stroke and its causes
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