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WO2017031342A1 - Biomarqueur circulant pour le syndrome de brugada - Google Patents

Biomarqueur circulant pour le syndrome de brugada Download PDF

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Publication number
WO2017031342A1
WO2017031342A1 PCT/US2016/047596 US2016047596W WO2017031342A1 WO 2017031342 A1 WO2017031342 A1 WO 2017031342A1 US 2016047596 W US2016047596 W US 2016047596W WO 2017031342 A1 WO2017031342 A1 WO 2017031342A1
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Prior art keywords
protein
subject
mespl
level
antibody
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Samuel C. Dudley
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Rhode Island Hospital
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Rhode Island Hospital
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • This invention relates to Brugada Syndrome.
  • aspects of the present subject matter feature a diagnostic/prognostic test for measuring circulating MESP1 (mesoderm posterior basic helix- loop-helix transcription factor 1) levels in a sample for Brugada Syndrome (BrS) diagnosis.
  • MESP1 mesoderm posterior basic helix- loop-helix transcription factor 1
  • test sample such as blood is obtained by venipuncture, and the sample comprises circulating cells such as white cells, monocytes, T-cells or a bodily fluid such as blood, serum, or plasma.
  • test sample comprises saliva.
  • test sample comprises a buffy coat fraction of blood.
  • the buffy coat is a standard fraction of an anti-coagulated blood sample that contains most of the white blood cells (WBCs) and platelets following density gradient centrifugation of the blood.
  • WBCs white blood cells
  • WBCs white blood cells
  • platelets following density gradient centrifugation of the blood.
  • subject is a human being characterized as comprising a risk of heart disease, e.g.
  • a diagnostic or prognostic level of an MESP1 protein or an MESP1 nucleic acid is a level that is decreased by at least 10% (at least 20%, 30%, 40%, 50% or more) compared to a normal control.
  • the subject has not been diagnosed as having a heart disease and has not had a cardiac event such as an arrhythmia, a heart valve disease or problem,
  • the subject has at least one grandparent, parent, sibling, or child who has been diagnosed with BrS .
  • the subject has at least one grandparent, parent, sibling, or child who has been determined to have a mutation in a sodium voltage-gated channel alpha subunit 5 (SCN5A), glycerol-3-phosphate dehydrogenase 1- like (GPD1L), calcium voltage-gated channel subunit alphal C (CACNA1C), calcium voltage- gated channel auxiliary subunit beta 2 (CACNB2), potassium voltage-gated channel subfamily E regulatory subunit 3 (KCNE3), potassium voltage-gated channel subfamily D member 3
  • SCN5A sodium voltage-gated channel alpha subunit 5
  • GPD1L glycerol-3-phosphate dehydrogenase 1- like
  • CACNA1C calcium voltage-gated channel subunit alphal C
  • CACNB2 calcium voltage- gated channel auxiliary subunit beta 2
  • KCNE3 potassium voltage-gated channel subfamily E regulatory subunit
  • KCND3 sodium voltage-gated channel beta subunit 1 (SCNIB), sodium voltage-gated channel alpha subunit 10 (SCNIOA), or hes related family bHLH transcription factor with YRPW motif 2 (HEY2) gene.
  • the subject is male.
  • the subject self- identifies as of Asian descent.
  • the subject has experienced syncope. In various embodiments, the subject has experienced syncope less than about 5, 4, 3, 2, or 1 times (e.g., has never experienced syncope). In some embodiments, the subject has had an electrocardiogram that did not reveal Brugada Syndrome (or an increased risk of Brugada Syndrome) and/or a normal electrocardiogram.
  • the subject has been diagnosed with asymptomatic Brugada Syndrome.
  • a significant advantage of the use of MESP1 as a biomarker includes the advantage of diagnostic or prognostic utility in a wide set of subtypes of Brugada Syndrome, e.g. , regardless of the genotype or expression level of other biomarkers for this disorder such as SCN5A and/or HuR.
  • the subject has not been determined to have an abnormality relating to SCN5A (e.g., a reduced full-length SCN5A protein level, a reduced full-length SCN5A mRNA level, an increased splice variant SCN5A protein level, or an increased splice variant SCN5A mRNA level or in a test sample, or a mutation in an SCN5A gene).
  • an abnormality relating to SCN5A e.g., a reduced full-length SCN5A protein level, a reduced full-length SCN5A mRNA level, an increased splice variant SCN5A protein level, or an increased splice variant SCN5A mRNA level or in a test sample, or a mutation in an SCN5A gene.
  • a subject has been tested for such a SCN5A abnormality, and the test was negative.
  • the subject does not have a grandparent, parent, sibling, or child who has been identified as having a SCN5A abnormality
  • Brugada Syndrome has been associated with reduced cardiac sodium channel current, in part because of a reduction in SCN5A mRNA abundance.
  • Reduced SCN5A contributes to the arrhythmic risk in heart failure.
  • the reduction in cardiac SCN5A mRNA abundance is reflected in circulating white cells that also express SCN5A.
  • a diagnostic or prognostic level of a short variant form of SCN5A e.g. , a splice variant
  • SCN5A e.g. , a splice variant
  • a diagnostic or prognostic level of a short variant form of SCN5A is a level that is decreased by at least 10% (at least 20%, 30%, 40%, 50% or more) compared to a normal control as well. The presence of the variants caused the reduced abundance of the full-length SCN5A mRNA.
  • the splice variant of the SCN5A gene is a splice variant produced from alternative splicing within Exon 28 of the SCN5A gene.
  • the splice variant is a SCN5A Exon 28 B splice variant (a.k.a., E28B), a SCN5A Exon 28 C splice variant (a.k.a., E28C), or a SCN5A Exon 28 D splice variant (a.k.a., E28D).
  • the presence of one or more SCNA splice variants E28B, E28C and/or E28D in the biological sample identifies the subject as being at risk for developing arrhythmia (methods and compositions of SCN5A splice variants, e.g. , SEQ ID NOs: 7, 8, and 9 of U.S. Patent Application Publication No. 2012/0129179, published May 24, 2012, and PCT International Patent Application Publication No. WO 2012/094651, published July 7, 2012 which are incorporated therein in their entireties).
  • the methods described herein may also include computing a level of an SCN5A variant, the cardiac transcription factor MEF2C (myocyte enhancer factor-2), or an HU protein (more preferably, HuR), or any combination thereof, e.g. , with a binding agent.
  • a binding agent comprise an antibody or a fragment thereof, a detectable protein or a fragment thereof (such as an MESP1 ligand), a nucleic acid molecule such as an oligonucleotide/polynucleotide comprising a sequence that is complementary to patient mRNA or a cDNA produced from patient mRNA, or any combination thereof.
  • the antibody may be
  • a detectable moiety e.g. , a fluorescent compound or a radioactive agent (e.g. , I).
  • a detectable moiety e.g. , a fluorescent compound or a radioactive agent (e.g. , I).
  • a detectable moiety e.g. , a fluorescent compound or a radioactive agent (e.g. , I).
  • the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine.
  • the antibody can also be detectably
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a specific binding agent describes agents having greater than 10-fold, preferably greater than 100-fold, and most preferably, greater than 1000-fold affinity for the target molecule as compared to another molecule.
  • specific is used to indicate that other biomolecules present in the sample do not
  • an antibody has a binding affinity in the low micromolar (10 "6 ), nanomolar (10 "7 -10 “9 ), with high affinity antibodies in the
  • the present subject matter provides a composition utilizing a binding agent, wherein the binding agent is attached to a solid support, (e.g. , a strip, a polymer, a bead, a nanoparticle, a plate such as a multiwell plate, or an array such as a microarray).
  • a solid support e.g. , a strip, a polymer, a bead, a nanoparticle, a plate such as a multiwell plate, or an array such as a microarray.
  • nucleic acid in a test sample may be amplified (e.g. , using PCR) before or after the nucleic acid to be measured is hybridized with the probe.
  • Various embodiments comprise reverse transcription polymerase chain reaction (RT-PCR) to detect mRNA levels.
  • RT-PCR reverse transcription polymerase chain reaction
  • the mRNA (or a portion thereof) in a test sample is converted to cDNA or partial cDNA and then the cDNA or partial cDNA is hybridized to a probe (e.g.
  • the solid support comprises a polymer, to which an agent is chemically bound, immobilized, dispersed, or associated.
  • a polymer support may be, e.g. , a network of polymers, and may be prepared in bead form (e.g. , by suspension polymerization).
  • the location of active sites introduced into a polymer support depends on the type of polymer support. For example, in a swollen-gel-bead polymer support the active sites are distributed uniformly throughout the beads, whereas in a macroporous-bead polymer support they are predominantly on the internal surfaces of the macropores.
  • the solid support e.g. , a device, may contain an MESP1 binding agent alone or together with a binding agent for at least one, two, three or more other molecules, e.g. , SCN5A.
  • the present subject matter provides diagnostic tests carried out using a bodily fluid or circulating cells such as nucleated blood cells.
  • the cells e.g. , white blood cells
  • the cells are lysed to yield a cell lysate prior to contacting the test sample (cell or cell lysate) with an MESP1 binding agent.
  • detection is accomplished using an enzyme- linked immunosorbent assay (ELISA) or Western blot format.
  • the binding agent comprises an MESP1 nucleic acid (e.g. , primers or probe that are complementary for MESP1 RNA or cDNA), and the detecting step is accomplished using a polymerase chain reaction (PCR) or Northern blot format, or other means of detection.
  • PCR polymerase chain reaction
  • a probe or primer is about 10-20, 15-25, 15-35, 15-25, 20-80, 50- 100, or 10- 100 nucleotides in length, e.g. , about 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 nucleotides in length or less than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 nucleotides in length.
  • a cell is lysed to release a protein or nucleic acid.
  • Numerous methods for lysing cells and assessing protein and nucleic acid levels are known in the art.
  • sample refers to a biological sample obtained for the purpose of evaluation in vitro.
  • the sample or patient sample preferably may comprise any body fluid.
  • the bodily fluid includes, but is not limited to, blood, plasma, serum, lymph, breast milk, saliva, mucous, semen, vaginal secretions, cellular extracts, inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, or urine obtained from the subject.
  • the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample.
  • the sample comprises blood or a fraction thereof (e.g.
  • the method further comprises repeating the providing, contacting, detecting, and computing steps over time.
  • a progressive decrease over time in the level of an MESP protein or MESP nucleic acid indicates a progressive worsening of Brugada Syndrome, or increased risk of sudden cardiac death (SCD).
  • the method may also include the step of treatment following risk stratification as described above.
  • a method of treatment is also within the invention.
  • a method of monitoring treatment of a patient with Brugada Syndrome by observing a change in level of an MESPl protein or an MESPl nucleic acid as described above.
  • an MESPl elevating medication is administered to the patient.
  • the MESPl elevating medication comprises an MESPl protein or fragment thereof, a nucleic acid encoding an MESPl protein or fragment thereof, e.g., using gene therapy.
  • Other medications that improve the condition of the patient identified using the risk stratification methods described herein include administration of an antiarrhythmic drug, implanted cardioverter-defibrillator (ICD), angiotensin converting enzyme inhibitor (ACE), angiotensin II receptor blocker, beta-blocker, digoxin, diuretic, blood vessel dilator, aldactone inhibitor, or calcium channel blocker.
  • ICD implanted cardioverter-defibrillator
  • ACE angiotensin converting enzyme inhibitor
  • angiotensin II receptor blocker beta-blocker
  • digoxin digoxin
  • diuretic diuretic
  • blood vessel dilator blood vessel dilator
  • aldactone inhibitor aldactone inhibitor
  • calcium channel blocker calcium channel blocker
  • the antiarrhythmic agent comprises any one of a group of pharmaceuticals that are used to suppress abnormal rhythms of the heart (cardiac arrhythmias), such as atrial fibrillation, atrial flutter, ventricular tachycardia, and ventricular fibrillation.
  • cardiac arrhythmias such as atrial fibrillation, atrial flutter, ventricular tachycardia, and ventricular fibrillation.
  • the anti-arrhythmic agent is a Singh Vaughan Williams (SVW) Class I, II, III, IV, or V anti-arrhythmic agent.
  • the antiarrhythmic agent is a SVW Class IA, IB, IC, or III anti-arrhythmic agent.
  • the antiarrhythmic agent may be a fast-channel blocker, a beta blocker, a slow channel blocker, a sodium channel blocking agent, a potassium channel blocking agent, or a calcium channel blocking agent.
  • the anti-arrhythmic agent in some aspects is one of Quinidine, Procainamide, Disopyramide, Lidocaine, Phenytoin, Mexiletine, Tocainide, Flecainide, Propafenone, Moricizine, Propranolol, Esmolol, Timolol, Metoprolol, Atenolol, Bisoprolol, Amiodarone, Sotalol, Ibutilide, Dofetilide, Dronedarone, E-4031 , Verapamil, Diltiazem, Adenosine, Digoxin, Ajmaline, Pilsicainide, or Magnesium Sulfate.
  • the SVW Class IA is Quinidine, Procainamide, or Disopyramide.
  • the SVW Class IB antiarrhythmic agent is Lidocaine, Phenytoin, Mexiletine, or Tocainide.
  • the SVW Class IC anti-arrhythmic agent is Flecainide, Propafenone, Moricizine, or Encainide.
  • the SVW Class III anti-arrhythmic agent is Dronedarone,
  • the anti-arrhythmic agent is NAD + or mitoTEMPO.
  • a kit comprising an MESPl binding agent and instructions for using the agent for evaluating arrhythmic risk or assessing the severity of heart failure.
  • the agent is attached to a solid support such a test strip.
  • the kit optionally contains buffers, enzymes, salts, stabilizing agents, preservatives, and a container for receiving a patient test sample of bodily fluid or cell.
  • a container contains an anticoagulant, cell separation agent (e.g. , to separate white cells from red blood cells), or a cell lysis agent, e.g.
  • kits comprising agents for measuring a group of markers, wherein the group of markers are defined as described in any of the preceding paragraphs, or panels containing figures, or other descriptions of preferred sets or panels of markers found herein. In some variations, such agents are packaged together. In some variations, the kit further includes an analysis tool for evaluating risk of an individual developing arrhythmia from measurements of the group of markers from at least one biological sample from the subject.
  • the diagnostic or prognostic assay is optionally formulated in a two-antibody binding format in which one MESPl protein- specific antibody captures an MESPl protein, in a patient sample and another MESPl -specific antibody is used to detect captured protein.
  • the capture antibody is immobilized on a solid phase, e.g. , an assay plate, an assay well, a nitrocellulose membrane, a bead, a dipstick, or a component of an elution column.
  • the second antibody i.e. , the detection antibody, is typically tagged with a detectable label such as a colorimetric agent or radioisotope.
  • the invention also describes diagnostic test system that obtains test results data representing levels of a marker in at least one biological sample.
  • the results are collected and tracked and a means for computing an index value from said marker, wherein the index value comprises an arrhythmic risk score or a heart failure risk score, and a means of reporting the index value.
  • the written report is electronic.
  • the written report is a printed paper report.
  • the present subject matter provides improvements over existing methodologies for, e.g. , diagnosing Brugada Syndrome, assessing the risk of arrhythmia or SCD, determining prognosis, and assessing Brugada Syndrome severity in a subject.
  • Some embodiments provide an improvement comprising: obtaining marker measurement data that is representative of measurements of at least two markers in a sample from the subject, and evaluating the risk of developing Brugada Syndrome in the subject based on an output from a model, wherein the model is executed based on an input of the biomarker measurement data.
  • an array of testing for Brugada Syndrome may include the combination of one or more methods disclosed herein with or without one or more methods known in the art for determining or evaluating Brugada Syndrome such as genetic testing, a pulmonary function test, an electrocardiography (ECG), an X-ray of the chest, an echocardiography, and/or the detection of an altered heart sound.
  • ECG electrocardiography
  • aspects of the present subject matter provide methods of treating a subject.
  • the subject has been diagnosed with Brugada Syndrome according to a method disclosed herein.
  • the subject is determined to be at risk of suffering from SCD.
  • Methods of monitoring treatment and assessing treatment efficacy are also provided.
  • Methods for reducing a subject's risk of suffering from SCD are also disclosed herein.
  • Embodiments of the present subject matter are useful for monitoring and evaluating the effectiveness of any treatment or candidate treatment for Brugada Syndrome.
  • methods disclosed herein may be used in a clinical setting to evaluate treatment progression or efficacy, and/or during a clinical trial to evaluate the effectiveness of a new therapy.
  • Therapies that improve the condition of the patient identified using the risk stratification methods described herein include administration of a drug or implantation of a cardioverter-defibrillator (ICD).
  • ICD cardioverter-defibrillator
  • Therapies for Brugada Syndrome and/or reducing the risk of SCD include administration of one or more antiarrhythmic agents.
  • an antiarrhythmic agent include any one of a group of pharmaceuticals that are used to suppress abnormal rhythms of the heart (cardiac arrhythmias), such as atrial fibrillation, atrial flutter, ventricular tachycardia, and ventricular fibrillation.
  • the anti-arrhythmic agent is a Singh Vaughan Williams (SVW) Class I, II, III, IV, or V anti- arrhythmic agent.
  • the antiarrhythmic agent is a SVW Class IA, IB, IC, or III anti-arrhythmic agent.
  • antiarrhythmic agent may be a fast-channel blocker, a beta blocker, a slow channel blocker, a sodium channel blocking agent, a potassium channel blocking agent, or a calcium channel blocking agent.
  • the anti- arrhythmic agent in some aspects is one of Quinidine, Procainamide, Disopyramide, Lidocaine, Phenytoin, Mexiletine, Tocainide, Flecainide, Propafenone,
  • the SVW Class IA is Quinidine, Procainamide, or Disopyramide.
  • the SVW Class IB antiarrhythmic agent is Lidocaine, Phenytoin, Mexiletine, or Tocainide.
  • the SVW Class IC antiarrhythmic agent is Flecainide, Propafenone, Moricizine, or Encainide.
  • the SVW Class III anti- arrhythmic agent is Dronedarone, Amiodarone, or Ibutilide.
  • the anti- arrhythmic agent is NAD+ or mitoTEMPO.
  • Various implementations of the present subject matter relate to a method of monitoring treatment of a patient with Brugada Syndrome by observing a change in level of a biomarker disclosed herein.
  • the amount of a treatment may be adjusted upon observation of a change in the level of a biomarker.
  • the level of one or more biomarkers disclosed herein is measured, e.g., at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or at least once every 1, 2, 3, 4, or 5 years, e.g.
  • the present subject matter also provides related methods of determining need for therapy or prophylaxis of a subject.
  • the present subject matter provides methods of determining need for therapy for Brugada Syndrome or prophylaxis for SCD for a subject identified as having a risk (e.g. , an increased risk compared to a normal control or other patients afflicted with Brugada Syndrome) for SCD.
  • the method comprises the step of detecting an abnormal level of a biomarker in a test sample from the subject, wherein an increased level indicates the need for a therapy for Brugada Syndrome or prophylaxis for SCD.
  • Intervention via active therapy or active prophylaxis may decrease the risk for developing the medical condition, disease, or syndrome.
  • Aspects of the present subject matter provide methods of decreasing the severity of Brugada Syndrome in a subject.
  • the methods may comprise (i) determining a level of a biomarker in a test sample from a subject, and (ii) administering to the subject a therapeutic or prophylactic agent, if the level determined in (i) is abnormal.
  • Aspects of the present subject matter also provide methods of decreasing the risk of SCD in a subject.
  • the method comprises the steps of (i) determining a level of a biomarker in a test sample from the subject, and (ii) administering to the subject a therapeutic or prophylactic agent, if the level determined in (i) is abnormal.
  • a method to monitor the efficacy of treatment comprises determining a level of a biomarker in a test sample of a subject before and after treatment.
  • a change in the level of the biomarker in the sample taken after treatment compared to the level of the biomarker before treatment indicates efficacy of the treatment.
  • a first test sample is obtained from the subject to be treated prior to initiation of therapy or part way through a therapy regime.
  • a test sample is provided to a person performing the assay.
  • a first test sample is obtained or provided from a subject known not to suffer from a condition being treated.
  • the second test sample is obtained or provided in a similar manner, but at a time following onset of therapy.
  • the second test sample in some embodiments, is obtained or provided at the completion of, or part way through therapy, provided that at least a portion of therapy takes place between the isolation of the first and second test samples.
  • an increase in the level of MESP1 in the second test sample e.g. , post-treatment
  • the level of MESPl in the first test sample e.g. , prior to treatment or from a subject known not to suffer from the condition being treated
  • an increase in the level of full-length SCN5A in the second test sample indicates a degree of effective therapy.
  • a decrease in the level of SCN5A splice variant in the second test sample (e.g. , post-treatment) compared to the level of SCN5A splice variant in the first test sample (e.g. , prior to treatment or from a subject known not to suffer from the condition being treated) indicates a degree of effective therapy.
  • Syndrome comprising (a) providing a test sample from a subject, wherein said test sample comprises a circulating cell or a bodily fluid; (b) assaying the level of a MESPl protein or MESPl protein-encoding mRNA in the test sample; and (c) diagnosing the subject with Brugada Syndrome if the level of the MESPl protein or MESPl protein-encoding mRNA is reduced in the test sample compared to a normal control.
  • the subject is diagnosed with Brugada Syndrome if the level of the MESPl protein or MESPl protein-encoding mRNA is decreased by least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 5-50%, or 50-75% in the test sample compared to a normal control.
  • the present subject matter also provides a method for treating Brugada Syndrome in a subject who has been diagnosed with Brugada Syndrome according to a method disclosed herein, comprising implanting an ICD into the subject.
  • Also provided is a method for identifying whether a subject who has Brugada Syndrome is at risk of suffering from SCD comprising (a) providing a test sample from the subject, wherein said test sample comprises a circulating cell or a bodily fluid; (b) assaying the level of a MESPl protein or MESPl protein-encoding mRNA in the test sample; (c) identifying the subject as at risk of suffering from SCD if the level of the MESPl protein or MESPl protein-encoding mRNA is reduced in the test sample compared to a normal control.
  • the subject is identified as at risk of suffering from SCD if the level of the MESPl protein or MESPl protein-encoding mRNA is decreased by least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 5-50%, or 50-75% in the test sample compared to a normal control.
  • the present subject matter also provides a method for treating Brugada Syndrome in a subject who has been identified as at risk of dying from SCD according to a method disclosed herein, comprising implanting an ICD into the subject.
  • the present subject matter further provides a method for monitoring the risk of SCD in a subject who has been diagnosed with Brugada Syndrome, comprising periodically determining the level of MESPl protein or MESPl protein-encoding mRNA in the subject, and (1) identifying the risk of SCD as increasing if the level of MESPl protein or MESPl protein- encoding mRNA in the subject decreases over time; (2) identifying the risk of SCD as decreasing if the level of the MESPl protein or MESPl protein-encoding mRNA in the subject increases over time; or (3) identifying the risk of SCD as neither increasing nor decreasing if the level of the MESPl protein or MESPl protein-encoding mRNA in the subject remains the same over time, wherein determining the level of the MESPl protein or MESPl protein-encoding mRNA in the subject comprises (a) providing a test sample from said subject, wherein said test sample comprises a circulating cell or a bodily fluid; and (b) assaying the level of the MESPl protein or M
  • the level of the MESPl protein or MESPl protein-encoding mRNA is determined at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. In certain embodiments, the level of MESPl protein or MESPl protein- encoding mRNA is determined at least about every 1, 2, 3, 4, 5, 8, 9, 10, 25, or 52 weeks or about once every 1, 2, 3, or 4 months.
  • Also included herein is a method for identifying whether a subject who comprises Brugada Syndrome is at risk of dying from SCD, comprising (a) providing a test sample from the subject, wherein the test sample comprises a circulating cell or a bodily fluid; (b) assaying the level of a MESPl protein or MESPl protein-encoding mRNA in the test sample; and (c) (1) comparing the level determined in (b) to a value in a database to identify the subject's absolute or relative risk of suffering from SCD, or (2) identifying the subject is at risk of suffering from SCD if the MESPl protein or MESPl protein-encoding mRNA is decreased by least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 5-50%, or 50-75% in the test sample compared to a normal control.
  • aspects of the present subject matter provide a method for identifying whether a therapy has improved Brugada Syndrome in a subject, comprising (a) providing a pre-therapy test sample from the subject; (b) assaying a pre-therapy level of a MESP1 protein or MESP1 protein- encoding mRNA in the pre-therapy test sample; (c) administering the therapy to the subject; (d) providing a post-therapy test sample from said subject, wherein said test sample comprises a circulating cell or a bodily fluid; (e) assaying a post-therapy level of the MESP1 protein or MESP1 protein-encoding mRNA in the post-therapy test sample; and (f) identifying the therapy as having improved Brugada Syndrome in the subject if the pre-therapy level of the MESP1 protein or MESP1 protein-encoding mRNA is lower than the post-therapy level of the MESP1 protein or MESP1 protein-encoding mRNA.
  • the therapy is a test therapy.
  • the subject assaying a
  • Methods of the present subject matter relating to assaying the level of MESP1 protein and/or mRNA may further include assaying the level of a SCN5A protein and/or mRNA, a HU protein and/or mRNA, and/or a MEF2C protein and/or mRNA.
  • the term “about” refers to any minimal alteration in the concentration or amount of an agent that does not change the efficacy of the agent in preparation of a formulation and in treatment of a disease or disorder (e.g. , Brugada Syndrome).
  • concentration range of the agents (e.g. , therapeutic/active agents) of the current disclosure also refers to any variation of a stated amount or range which would be an effective amount or range. Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value.
  • the antibody is a polyclonal antisera or monoclonal antibody.
  • the invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g. , a Fab or (Fab)2 fragment; an engineered single chain FV molecule; or a chimeric molecule, e.g. , an antibody which contains the binding specificity of one antibody, e.g. , of murine origin, and the remaining portions of another antibody, e.g. , of human origin.
  • the invention further comprises a humanized antibody, wherein the antibody is from a non-human species, whose protein sequence has been modified to increase their similarity to antibody variants produced naturally in humans.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non- human amino acid residues are referred to herein as "import" residues, which are typically taken from an "import" antibody domain, particularly a variable domain.
  • transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • a “diagnostic test system,” means a system for obtaining test results data representing levels of multiple markers in at least one biological sample; means for collecting and tracking test results data for one or more individual biological samples; means for computing an index value from marker measurement data, wherein said biomarker measurement data is
  • the index value is arrhythmia risk score.
  • the arrhythmia risk score is computed according to the methods described herein for computing such scores.
  • the means for collecting and tracking test results data representing for one or more individuals comprises a data structure or database.
  • the means for computing arrhythmic risk score comprises a computer or microprocessor, comprising a visible display, an audio output, a link to a data structure or database, or a printer.
  • evaluating arrhythmic risk or “assessing the severity of heart failure” is used to indicate that the method according to the present invention will alone or together with other variables, (e.g., administration of an antiarrhythmic compound), establish or confirm the absence or presence of arrhythmia, or aid the physician in the prognosis, and or the monitoring of treatment.
  • variables e.g., administration of an antiarrhythmic compound
  • any such evaluation or assessment is made in vitro.
  • the patient sample is discarded afterwards.
  • the patient sample is solely used for the in vitro diagnostic method of the invention and the material of the patient sample is not transferred back into the patient's body.
  • the sample is a liquid sample, e.g., whole blood, serum or plasma.
  • fluoroimmunoassay an assay that has an agent labeled with a fluorophore.
  • isolated nucleic acid is meant a nucleic acid that is free of the genes which flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived.
  • the term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
  • PCR polymerase chain reaction
  • Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.
  • the isolated nucleic acid is a purified cDNA or RNA polynucleotide.
  • Isolated nucleic acid molecules also include messenger ribonucleic acid
  • cDNA (mRNA) molecules. cDNA is not naturally occurring.
  • mass spectrometry is meant an analytical technique that helps identify the amount and type of components present in a sample by measuring the mass-to-charge ratio and the abundance of gas-phase ions.
  • Exemplary techniques comprise electrospray ionization (ESI), matrix assisted laser desorption (MALDI), MALDI-TOF (Time of flight), Fourier transform ion cyclotron resonance (FTIC), and surface-enhanced laser desorption (SELDI).
  • modulate means regulating or adjusting to a certain degree.
  • normal amount refers to a normal amount of a complex in an individual known not to be diagnosed with arrhythmia or heart failure.
  • the amount of the protein can be measured in a test sample and compared to the "normal control level," utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cutoff points and abnormal values (e.g. , for arrhythmia).
  • the normal control level means the level of one or more proteins or combined protein indices typically found in a subject known not suffering from arrhythmia. Such normal control levels and cutoff points may vary based on whether a protein is used alone or in a formula combining with other proteins into an index. Alternatively, the normal control level can be a database of protein patterns from previously tested subjects who did not convert to arrhythmia over a clinically relevant time horizon.
  • the level that is determined may be the same as a control level or a cut off level or a threshold level, or may be increased or decreased relative to a control level or a cut off level or a threshold level.
  • the control subject is a matched control of the same species, gender, ethnicity, age group, smoking status, body mass index (BMI), current therapeutic regimen status, medical history, or a combination thereof, but differs from the subject being diagnosed in that the control does not suffer from the disease in question or is not at risk for the disease.
  • the level that is determined may an increased level.
  • the term "increased" with respect to level refers to any % increase above a control level.
  • the increased level may be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85% increase, at least or about a 90% increase, at least or about a 95% increase, relative to a control level.
  • the level that is determined may a decreased level.
  • the term "decreased" with respect to level refers to any % decrease below a control level.
  • the decreased level may be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, at least or about a 95% decrease, relative to a control level.
  • nucleic acid refers to polynucleotides such as
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • Polynucleotides, polypeptides, or other agents are purified and/or isolated.
  • an "isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purified compounds are at least 60% by weight (dry weight) the compound of interest.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest.
  • a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.
  • a purified or isolated polynucleotide ribonucleic acid (RNA) or
  • concentrations of agents using an antibody by use of labeled antigen (e.g., gamma-radioactive isotopes of iodine).
  • labeled antigen e.g., gamma-radioactive isotopes of iodine
  • Receiver-operating characteristics describe the accuracy of a diagnostic method (See, Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-577).
  • the ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed (also called a Youen's J Statistic or the
  • Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1-specificity for the complete range of decision thresholds.
  • On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true-positive test results)/(number of true-positive+number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the false-positive fraction On the x-axis is the false-positive fraction, or 1-specificity [defined as (number of false-positive results)/(number of true-negative+number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity/ 1-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes (if the ROC plot falls completely below the 45°diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than” to "less than” or vice versa).
  • the closer the plot is to the upper left corner the higher the overall accuracy of the test.
  • One preferred way to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the conversion to arrhythmia or heart failure, and can mean a subject's "absolute” risk or “relative” risk.
  • a high risk subject may comprise a subject at risk of developing arrhythmia or heart failure within 1 year.
  • a high risk subject comprises a subject at risk of suffering from SCD.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1-p) is the probability of no event) to no-conversion.
  • salt refers to acid or base salts of the agents used herein.
  • acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid, and the like) salts, and quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
  • the severity of a disease may be expressed in terms of severity of symptoms that may be mild, severe or life-threatening. Common symptoms that are considered comprise chest pain, fainting, light-headedness, dizziness, paleness, shortness of breath, or sweating.
  • arrhythmia may be quantified using a scoring method. For example, scores may correlate the incidences of ventricular fibrillation, ventricular tachycardia, and ventricular premature beats in early myocardial ischemia.
  • subject includes all members of the animal kingdom prone to suffering from the indicated disorder.
  • subject includes all members of the animal kingdom that suffer from or may suffer from the indicated disorder.
  • the subject is a mammal, and in some aspects, the subject is a human.
  • companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs, and other domesticated and wild animals.
  • substantially pure is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it.
  • the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.
  • an isolated or purified cell is one that has been substantially separated or purified away from other biological components of the organism in which the cell naturally occurs, such as other cells of the organism.
  • an isolated lymphocyte cell population is a population of lymphocytes that is substantially separated or purified away from other blood cells, such as red blood cells.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and recovery (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • compositions and methods for treating a disease or disease state it is understood that the term “prevent” does not require that the disease state (e.g. , Brugada Syndrome) be completely thwarted.
  • the term “prevent” can encompass partial effects when the agents disclosed herein are administered as a prophylactic measure.
  • the prophylactic measures include, without limitation, administration to one (or more) individual(s) who is suspected of being diagnosed with, e.g., Brugada Syndrome.
  • assaying means using an analytic procedure to qualitatively assess or quantitatively measure the presence or amount or the functional activity of a target entity.
  • assaying the level of a compound means using an analytic procedure (such as an in vitro procedure) to qualitatively assess or
  • phrases such as "at least one of or "one or more of may occur followed by a conjunctive list of elements or features.
  • the term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features.
  • the phrases “at least one of A and ⁇ ;” “one or more of A and ⁇ ;” and “A and/or B” are each intended to mean "A alone, B alone, or A and B together.”
  • a similar interpretation is also intended for lists including three or more items.
  • the phrases "at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible
  • a small molecule is a compound that is less than 2000 daltons in mass.
  • the molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons.
  • BrS is an inherited sudden death condition caused mostly by reductions in cardiac sodium current. The condition arises when sodium channels behave abnormally, wherein the movement of sodium ions into the cells is restricted which results in changes in the ECG
  • SCN5A was the first gene known to be associated with BrS. About 20% of BrS patients are thought to carry mutations in SCN5A gene and mutations in more than 10 genes have been reported to cause BrS. Nevertheless, approximately 80% of clinically diagnosed BrS patients do not carry any mutation known to cause BrS. Sodium channel transcript levels have been shown to correlate between heart and white cells, indicating that white cells are useful to assess cardiac SCN5A transcription. Therefore, measurement of white blood cell SCN5A expression levels may reveal cardiac sodium channel expression changes in BrS patients.
  • ICD implantable cardioverter defibrillator
  • the EP study is a 1-2 hour procedure that allows your doctor to assess the electrical system in the heart using special types of wires that are placed in the heart to sense the heart's activity and pace the heart when needed.
  • the EP study is used to identify the type of abnormal rhythm and determine the effect of certain medications. No treatments are performed in the EP lab, but the study may provide important information, such as your risk for passing out or possibly dying from an arrhythmia. If the doctor schedules an EP test, it is important to discuss what medications being taking. Some medications must be stopped anywhere from 24 hours to a week prior to the EP study.
  • An ICD is an implantable device about the size of a small pager.
  • An ICD can detect and prevent the kind of arrhythmias responsible for causing patients to faint or die. If the ICD detects this type of arrhythmia, it sends energy to the heart to "shock" it back to a normal rhythm.
  • Several grave complications and risks arise when having an ICD including unnecessary electrical pulses or shocks that aren't needed, a damaged wire or a very fast heart rate due to extreme physical activity may trigger unnecessary pulses. These pulses also can occur if after forgetting to take the prescribed medicines. Children tend to be more physically active than adults, and thus younger people who have ICDs are more likely to receive unnecessary pulses than older people. Pulses sent too often or at the wrong time can damage the heart or trigger an irregular, sometimes dangerous heartbeat. People who have ICDs may be at higher risk for heart failure.
  • the invention further comprises a subject's risk for arrhythmia.
  • arrhythmia is synonymous with "cardiac dysrhythmia” or “cardiac arrhythmia” and refers to any condition in which there is abnormal electrical activity in the heart.
  • the cardiac arrhythmia may be a ventricular arrhythmia, such as ventricular fibrillation, ventricular tachycardia, or an arrhythmic condition in which both ventricular fibrillation and ventricular tachycardia are present.
  • the cardiac arrhythmia may be an atrial arrhythmia, e.g., an atrial fibrillation, atrial tachycardia, or an arrhythmic condition in which both atrial fibrillation and atrial tachycardia are present. Other types of cardiac arrhythmias are described below.
  • the cardiac arrhythmia may be characterized by an abnormal heart rate, such as a bradycardia or a tachycardia.
  • a bradycardia is a cardiac arrhythmia in which the resting heart rate is slower than normal, and is characterized by a resting heart rate in an adult human which is slower than 60 beats per minute.
  • the bradycardia is a sinus bradycardia caused by sinus arrest or AV (atrioventricular) block or heart block.
  • the bradycardia is caused by a slowed electrical conduction in the heart.
  • a tachycardia is a cardiac arrhythmia in which the resting heart rate is faster than normal, which is faster than 100 beats per minute.
  • the tachycardia may be a sinus tachycardia, and is not caused by physical exercise, emotional stress, hyperthyroidism, ingestion or injection of substances, such as caffeine or amphetamines.
  • the tachycardia may not be a sinus tachycardia, e.g., a tachycardia resulting from automaticity, reentry (e.g., fibrillation), or triggered activity.
  • the tachycardia may be caused by a slowed electrical conduction in the heart, an ectopic focus, or is combined with abnormal rhythm.
  • SCD sudden cardiac death
  • SCD is a sudden, unexpected death caused by loss of heart function (sudden cardiac arrest).
  • SCD is not a heart attack (myocardial infarction) but can occur during a heart attack.
  • SCD occurs when the electrical system to the heart malfunctions and suddenly becomes very irregular (i.e., there is an arrhythmia).
  • the most common life-threatening arrhythmia is ventricular fibrillation, which is an erratic, disorganized firing of impulses from the ventricles (the heart's lower chambers).
  • MESP1 (Mesoderm posterior protein 1) basic helix loop helix
  • MESP1 is a cardiac specific transcription factor expressed in white blood cells MESP1 has been considered the cardiac master regulator, driving cardiovascular specification and inhibiting other mesodermal lineages. MESP1 is expressed in the nascent mesoderm but is abruptly downregulated as the newly formed mesoderm migrates out of the primitive streak. MESP1 expressing cells were found to give rise to all cardiac lineages.
  • Exemplary regions or fragments of MESPl include residues 83-140 (helix-loop-helix domain), residues 83-122 (DNA binding region), 97-140 (dimerization interface), region 16.- 167, and region 182-185.
  • Cardiac voltage-gated Na + (Nav) channels consist of a heteromeric assembly of pore- forming a subunit and auxiliary ⁇ subunits that modulate channel functions.
  • Nav 1.5 (SCN5A) is the major Nav a subunit expressed in the mammalian myocardium, whereas multiple Nav ⁇ subunits have been described in cardiomyocytes.
  • Voltage-gated Na + channels play a critical role in the membrane excitability of cardiomyocytes by generating the rapid upstroke of the action potential. Additionally, Nav channels govern the impulse conduction velocity in the
  • the splice variant is a SCN5A Exon 28 B splice variant (a.k.a., E28B; Exon 28 shown as SEQ ID NO: 3), a SCN5A Exon 28 C splice variant (a.k.a., E28C; Exon 28 shown as SEQ ID NO: 4), or a SCN5A Exon 28 D splice variant (a.k.a., E28D; Exon 28 shown as SEQ ID NO: 5).
  • the level may be an expression level of a full length transcript of SCN5A gene or of a splice variant of the SCN5A gene. Suitable methods of determining expression levels of transcripts of a gene are include direct methods of determining levels of transcripts (e.g., quantitative PCR) and indirect methods of determining levels of transcripts (e.g., Western blotting for translated protein or peptide products of the transcripts).
  • the level may be an activity level of a full-length transcript of the SCN5A gene that is determined via measurement, e.g., measurement of the sodium current.
  • E28B SCN5A Splice Variant Complete Amino Acid Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
  • E28C SCN5A Splice Variant Complete Amino Acid Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
  • E28D SCN5A Splice Variant Complete Nucleotide Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
  • E28D SCN5A Splice Variant Complete Amino Acid Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
  • SEQ ID NO: 11 A full-length SCN5A amino acid sequence is shown below.
  • Exemplary regions or fragments of SCN5A include residues 159-412 (ion transport region), 159-178 (transmembrane region), 842-862 (transmembrane region), and 1201-1224 (sodium ion transport-associated region).
  • Exemplary regions or fragments of SCN5A include residues 95-1022 (transmembrane region), 1563-1565 (phosphorylation site), 1731-1733 (methylation site), and 5172-5240 (transmembrane region).
  • HU proteins are RNA-binding proteins involved in diverse biological processes, such as neuronal development and cellular stress response.
  • the "H” stands for histone and "U” stands for the 93U strain used initially to isolate the E. coli ⁇ Escherichia coli) nucleoid.
  • HU is a small, basic, and thermostable dimeric DNA-binding protein, and is a major structural component of the nucleoid.
  • HU proteins affect the expression of their regulons through diverse mechanisms, from splicing to translation, e.g., their ability to stabilize target mRNA by binding to AREs in their 3' untranslated regions.
  • HU proteins recognize and bind to AU-rich RNA elements (AREs) and also show an empirical preference for U-rich sequences as well as some other RNA sequences.
  • AREs AU-rich RNA elements
  • Each HU protein has three RNA recognition motifs (RRMs 1-3), which share more than 90% amino acid sequence identity among family members.
  • RRMs 1-3 RNA recognition motifs
  • the SCN5A mRNA 3' untranslated region (UTR) was shown to contain two sets of AU-rich elements (ARE), which may be able to bind RNA-binding proteins such as HU proteins.
  • RBPs may regulate the expression of multiple mRNAs that encode functionally related proteins, termed RNA operons. Individual mRNAs can be members of multiple operons, forming higher-order "RNA regulons.”
  • HU proteins may perform their overall biological functions by coordinately regulating functionally related mRNAs. All of the biological functions of HU proteins are believed to be a result of their ability to bind specific target mRNAs and affect their expression. In the cytoplasm, HU proteins are best known for stabilizing target mRNAs, (for example, GAP-43, VEGF, and GLUT1), by binding to AREs in their 3' untranslated regions (UTRs) and prevent their degradation, and thus indirectly enhancing protein production.
  • target mRNAs for example, GAP-43, VEGF, and GLUT1
  • HU proteins are best known for stabilizing mRNAs, they can also affect target protein expression at the level of translation. Unlike their role in mRNA stability in which target protein expression is enhanced, HU proteins may act as enhancers or repressors of translation. HU proteins upregulate the translation of many target mRNAs, which result in increased recruitment of target mRNAs to polysomes, indicating increased translation initiation. In the nucleus, HU proteins serve as regulators of polyadenylation and alternative splicing.
  • HuA Human in Human
  • HuB HelNl in Human
  • HuC HuD
  • HuD mammalian RNA-binding proteins
  • HuR GenBank accession number: NM_001419.2
  • HuB GenBank accession number: NM_004432.3
  • HuC ELAVL3, GenBank accession number: NM_001420.3
  • HuD ELAVL4, GenBank accession number: NM_001144774.1
  • HuR The family HU family member, HuR, has a variety of biological functions. Through its post-transcriptional regulation of targets, such as several genes controlling cell growth and proliferation, HuR is believed to mediate cellular response to DNA damage and other types of stress.
  • HuR contains three RNA-recognition motif (RRM) domains.
  • the RRM1 domain comprises residues 20-98 (79 amino acids in length);
  • RRM2 domain comprises residues 106-186 (81 amino acids in length);
  • the RRM3 domain comprises residues 244-322 (79 amino acids in length).
  • the protein is characterized by the following amino acid modifications:
  • This encoded protein contains 3 RNA-binding domains and binds cis-acting AU-rich elements. It stabilizes mRNAs and thereby regulates gene expression.
  • HuR GenBank Accession NPJ301410.2 (GI: 38201714), incorporated herein by reference.
  • Exemplary landmark residues, domains, or fragments of HuR include residues 243-326 (the RNA recognition motif 3 in vertebrate Hu-antigen R), residues 180-183 ( ⁇ -strand region), residues 157-167 (helical region), resiudes 146-156 ( ⁇ -strand region), residues 141-143
  • Exemplary regions or fragments of HuR nucleic acid sequence include bases 771-773 (phosphorylation site), bases 2332-2337 (poly A signal sequence), bases 6034-6039 (poly A signal sequence).
  • Exemplary regions or fragments of HuB include residues 38-115 (RNA recognition motif 1), 40-115 (RNA binding site), 120-209 (RNA recognition motif 2), 126-203 (putative RNA binding site), 239-251 (splicing variant), and domain 276-354.
  • HuB nucleic acid sequences include bases 936-938 (phosphorylation site), and 3787-3792 (regulator sequence, polyA signal sequence).
  • MEF2C Myocyte-specific enhancer factor 2C
  • the process of differentiation from mesodermal precursor cells to myoblasts has led to the discovery of a variety of tissue-specific factors that regulate muscle gene expression.
  • the myogenic basic helix-loop-helix proteins including yoD, myogenin, Myf-5, and M F4, are one class of identified factors.
  • a second family of DNA binding regulatory proteins is the myocyte-specific enhancer factor-2 (MEF-2) family. Each of these proteins binds to the MEF-2 target DNA sequence present in the regulatory regions of many muscle-specific genes.
  • Mef2 proteins regulate the sters-response during cardiac hypertrophy and tissue remodeling in cardiac and skeletal muscle.
  • MEF2C GenBank Accession NP_001180279.1 (GI: 301069386), incorporated herein by reference.
  • Exemplary regions or fragments of MEF2C include residues 2-38 (DNA binding site), 3- 57 (binding domain), 4-31 (compositionally biased region, lysine rich region), 21-73
  • Exemplary regions or fragments of MEF2C nucleic acid sequences include bases 331- 333 (upstream in-frame stop-codon), 484-687 (exon), and 1364- 1499 (exon).
  • antibody refers to immunoglobulin molecules
  • immunoglobulin (Ig) molecules i.e. , molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a b, F a b' and F( a b )2 fragments, and an F a b expression library.
  • specifically bind or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e. , bind) with other polypeptides or binds at much lower affinity (K d > 10 "6 ) with other polypeptides.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • J Fundamental Immunology Ch. 7
  • D variable region of about 10 more amino acids.
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
  • Certain classes have subclasses as well, such as IgGi, IgG 2 , and others.
  • the light chain may be a kappa chain or a lambda chain.
  • antigen-binding site or "binding portion” refers to the part of the
  • the antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light (“L”) chains.
  • V N-terminal variable
  • L heavy
  • FR framework regions
  • the term "FR” refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface.
  • epitopope includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor.
  • epitopope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 ⁇ ; preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495.
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include a full length protein or a fragment thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see pp. 59-103 in Goding (1986) Monoclonal Antibodies: Principles and Practice Academic Press).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • the antibodies to an epitope for an interested protein as described herein or a fragment thereof are humanized antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. 1986. Nature 321:522-525; Riechmann et al. 1988. Nature 332:323-329; Presta. 1992. Curr. Op. Struct. Biol. 2:593-596).
  • Humanization can be essentially performed following methods of Winter and coworkers (see, e.g., Jones et al. 1986. Nature 321:522-525; Riechmann et al. 1988. Nature 332:323-327; and Verhoeyen et al. 1988. Science 239: 1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such humanized antibodies are chimeric antibodies (e.g., U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the
  • the antibodies to an epitope of an interested protein as described herein or a fragment thereof are human antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter. 1991. J. Mol. Biol. 227:381-388; Marks et al. 1991. J. Mol. Biol. 222:581-597) or the preparation of human monoclonal antibodies (e.g., Cole et al. 1985. Monoclonal Antibodies and Cancer Therapy Liss; Boerner et al. 1991. J. Immunol. 147(l):86-95).
  • Exemplary antibodies against HuR (ELAVL1) protein include, but are not limited to, antibodies obtained from "antibodies online” (e.g., Cat. No. ABIN577055; Cat. No.
  • Exemplary antibodies against HuB protein include, but are not limited to, antibodies obtained from Sigma-Aldrich (e.g., H1538), antibodies obtained from LifeSpan Biosciences (e.g., LS-B9943, and more can be found at its website www.lsbio.com), antibodies obtained from Santa Cruz Biotech (e.g., sc-5982), any commercially available antibodies against HuB, and any antibodies that are generated by known method in the art utilizing the full-length protein or a fragment of human HuB (e.g., residues 38-115, residues 40-115, residues 120-209, residues 126- 203, residues 239-251, residues 276-354, any fragment or full length of SEQ ID NO: 16).
  • Sigma-Aldrich e.g., H1538
  • LifeSpan Biosciences e.g., LS-B9943, and more can be found at its website www.lsbio.com
  • Santa Cruz Biotech e.g., sc-5982
  • Exemplary antibodies against human SCN5A protein include, but are not limited to, antibodies obtained from "Thermo Scientific online” (e.g., Cat. No. PA5-34190; Cat. No. MA1- 27429; Cat. No. PA5-39462; Cat. No.
  • PA5-36074 PA5-36074, and more can be found at its website www.pierce-antibodies.com
  • antibodies obtained from "abcam.com” e.g., ab53724, ab62388, abl 16706, ab86321, and more can be found at its website www.abcam.com
  • antibodies obtained from Santa Cruz Biotech e.g., sc-271255, sc-81631, sc22758, sc23174, and more can be found at its website www.scbt.com
  • any commercially available antibodies against SCN5A and any antibodies that are generated by known method in the art utilizing the full-length protein or a fragment of human SCN5A (e.g., residues 159-412, residues 159-178, residues 842-862, residues 1201-1224, any fragment or full length of SEQ ID NO: 12).
  • Exemplary antibodies against human MEF2C protein include, but are not limited to, antibodies obtained from "Thermo Scientific online” (e.g., Cat. No. MA5-17119; Cat. No. PA5- 28247; Cat. No. PA5-34581; Cat. No.
  • PA5- 13287 PA5- 13287, and more can be found at its website www.pierce-antibodies.com
  • antibodies obtained from "abcam.com” e.g., ab64644, abl97070, abl91428, and more can be found at its website www.abcam.com
  • antibodies obtained from Santa Cruz Biotech e.g., sc-365862, sc- 13266, sc- 13268, and, more can be found at its website www.scbt.com
  • any commercially available antibodies against MEF2C and any antibodies that are generated by known method in the art utilizing the full-length protein or a fragment of human MEF2C (e.g., residues 2-38, residues 3-57, residues 4-31, residues 21-73, residues 87-134, residues 107-134, residues 110-156, residues 271-278, residues 368-399, any fragment or full length of SEQ ID NO: 18).
  • nucleic acid e.g. , an mRNA molecule
  • the nucleic acid Prior to amplification and/or detection of a nucleic acid (e.g. , an mRNA molecule), the nucleic acid is optionally purified from the samples. Alternately, samples can simply be directly subjected to amplification or detection, e.g. , following aliquotting and/or dilution.
  • Suitable primers to be used with the invention can be designed using any suitable method. It is not intended that the invention be limited to any particular primer or primer pair.
  • primers can be designed using any suitable software program, such as
  • LASERGENE® taking account of publicly available sequence information. Sequences for mRNA transcripts of MESP1, SCN5A, HU, and MEF2C proteins are publicly available. Thus, suitable amplification primers can be constructed based on well understood base-pairing rules. The presence of any mRNA or amplicon thereof can be detected, e.g. , by hybridization (e.g. , array or probe hybridization), amplification (e.g. , comprising RT-PCR), sequencing, and the like (as well as combinations of these or other approaches).
  • a pair of primers that is complementary to a single transcript is used.
  • one or more of the primers used comprises a sequence of nucleic acids that is not complementary to the transcript.
  • a primer may comprise a tag sequence that identifies the primer, and/or that may be used to identify the sample or amplicons derived from the sample after future processing (e.g. when multiple samples are analyzed using a single microarray).
  • the tag sequence is at least about 4, 5, 6, 7, 8, 9, or 10 nucleotides long and/or less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides long.
  • the primers of the invention are radiolabeled, or labelled by any suitable means (e.g. , using a non-radioactive fluorescent tag), to allow for rapid visualization of differently sized amplicons following an amplification reaction without any additional labelling step or visualization step.
  • the primers are not labelled, and the amplicons are visualized following their size resolution, e.g. , following agarose or acrylamide gel electrophoresis.
  • ethidium bromide staining of the PCR amplicons following size resolution allows visualization of the different size amplicons.
  • the primers of the invention be limited to generating an amplicon of any particular size.
  • the primers used to amplify the mRNA herein are not limited to amplifying the entire mRNA, or any subregion thereof.
  • the primers can generate an amplicon of any suitable length for detection.
  • amplification produces an amplicon at least 20 nucleotides in length, or alternatively, at least 50 nucleotides in length, or
  • At least 100 nucleotides in length or alternatively, at least 200 nucleotides in length.
  • Amplicons of any size can be detected using various technologies described herein and known in the art. Differences in base composition or size can be detected by conventional methods such as electrophoresis.
  • PCR detection using dual-labelled fluorogenic oligonucleotide probes commonly referred to as "TaqManTM” probes is used.
  • These probes are composed of short (e.g. , 20-25 base) oligodeoxynucleotides that are labelled with two different fluorescent dyes. On the 5' terminus of each probe is a reporter dye, and on the 3' terminus of each probe a quenching dye is found.
  • the oligonucleotide probe sequence is complementary to an internal target sequence present in a PCR amplicon. When the probe is intact, energy transfer occurs between the two fluorophores and emission from the reporter is quenched by the quencher by FRET.
  • TaqManTM probes are oligonucleotides that have a label and a quencher, where the label is released during amplification by the exonuclease action of the polymerase used in amplification. This provides a real time measure of amplification during synthesis.
  • a variety of TaqManTM reagents are commercially available, e.g.
  • Array-based detection can be performed using commercially available arrays, e.g. , from Affymetrix (Santa Clara, Calif.) or other manufacturers. Reviews regarding the operation of nucleic acid arrays include Sapolsky et al., 1999 Genet Anal: Biomolec Engin 14: 187- 192; Lockhart, 1998 Nature Medicine 4: 1235- 1236; Fodor, 1997a FASEB Journal 11 :A879; Fodor, 1997b Science 277: 393-395 and Chee et al., 1996 Science 274:610-614.
  • Array based detection is one exemplary method for identification nucleotides (such as mRNA molecules or amplicons thereof) in samples, due to the inherently high-throughput nature of array based detection.
  • probe arrays have been described in the literature and can be used to detect mRNA or amplicons thereof (e.g. , obtained using RT-PCR).
  • DNA probe array chips or larger DNA probe array wafers may be used.
  • DNA probe array wafers may comprise, e.g. , glass wafers on which high density arrays of DNA probes (short segments of DNA) have been placed. Each of these wafers can hold, for example, approximately 60 million DNA probes that are used to recognize longer sample DNA sequences (e.g. , from individuals or populations).
  • the recognition of a polynucleotide by the set of DNA probes on the glass wafer takes place through DNA hybridization. When a polynucleotide hybridizes with an array of DNA probes, the sample binds to those probes that are complementary to the polynucleotide sequence.
  • two DNA samples may be differentially labelled and hybridized with a single set of the designed genotyping arrays. In this way two sets of data can be obtained from the same physical arrays.
  • Labels that can be used include, but are not limited to, cychrome, fluorescein, or biotin (later stained with phycoerythrin-streptavidin after hybridization). Two- colour labelling is described in U.S. Patent No. 6,342,355, issued January 29, 2002. Each array may be scanned such that the signal from both labels is detected simultaneously, or may be scanned twice to detect each signal separately.
  • intensity data is collected by the scanner for all the markers for each of the individuals that are tested for presence or level of the marker.
  • the measured intensities are a measure indicative of the amount of a particular marker present in the sample for a given individual (i.e., the expression level).
  • Example 1 mRNA expression of MESPl in WBCs is decreased in both SCN5A(-) patients and SCN5A(+) patients
  • RNA samples were collected in BD vacutainer 9NC 0.129M (BD Biosciences). White blood cells (WBCs) were separated using Lympholyate- H sterile liquid. Total RNA was isolated using Trizol® (Life Technologies) and Direct-ZolTM RNA MiniPrep Kit and was reverse transcribed with QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative PCR (Q-PCR) analysis was performed using 7500-Fast Real Time PCR Systems with MESPl specific primers and probes and TaqMan® Gene Expression Master Mix (Applied Biosystems).
  • MESPl expression level was normalized by GAPDH (glyceraldehyde 3-phosphate). Differences between the groups were examined by one way ANOVA (Analysis of Variance) and t-tests. Results with P ⁇ 0.05 were considered statistically significant in all analysis.
  • the diagnostic odds ratio (DOR) of MESPl for BrS diagnosis was 11.96 (95% CI: 5.79- 24.73).
  • the assessment of the mRNA levels in blood SCN5A, MEF2C and HuR were useful for predicting BrS patients with an SCN5A mutation.
  • MESPl is used as a biomarker for BrS diagnosis and plays a roll in the pathophysiology of BrS.
  • Example 2 In vitro assays using an MESPl transcript hybridized to an isolated nucleic acid sequence
  • a method of identifying presence of Brugada Syndrome in a patient comprises analyzing a biological sample obtained from the subject, and detecting the expression of MESPl relative to a control.
  • the method further comprises providing RNA from WBC of the subject, reverse transcribing the isolated RNA to generate cDNA of MESPl, amplifying the cDNA with primers having nucleic acid sequences complementary to the target gene.
  • the primers are conjugated to a biological or chemical probe, and detecting a signal from the probe. Identification of a reduction in MESPl indicates the presence of Brugada Syndrome and the need for implanted defibrillation.

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Abstract

Cette invention se rapporte à un test diagnostique mesurant les transcrits géniques ou les protéines MEPSI circulantes dans un échantillon sanguin comme biomarqueur pour le Syndrome de Brugada.
PCT/US2016/047596 2015-08-20 2016-08-18 Biomarqueur circulant pour le syndrome de brugada Ceased WO2017031342A1 (fr)

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