WO2017031342A1 - Circulating biomarker for brugada syndrome - Google Patents

Circulating biomarker for brugada syndrome 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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PCT/US2016/047596
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French (fr)
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Samuel C. Dudley
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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

This invention relates to a diagnostic test measuring circulating MESPl proteins or gene transcripts in a blood sample as a biomarker for Brugada Syndrome.

Description

CIRCULATING BIOMARKER FOR BRUGADA SYNDROME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of priority to U.S. Provisional Application No.
62/207,854, filed on August 20, 2015, the entire content of which is incorporated herein by reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with government support under R01 HL 1024025, P01
HL058000, R01 HL106592, Veteran Administration Merit Award, R41 HL112355, and P20 GM 103652 awarded by National Institute of Health (NIH). The government has certain rights in the invention.
FIELD OF THE INVENTION
This invention relates to Brugada Syndrome.
REFERENCE TO THE SEQUENCE LISTING
The content of the text file named "21486-625001WO_Sequence_Listing.txt" which was created on August 18, 2016 and is 125 kilobytes in size is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Brugada Syndrome (BrS) is an inherited sudden death condition caused mostly by reductions in cardiac sodium current. The condition leads to a high incidence of sudden death in patients with structurally normal hearts. The syndrome typically manifests during adulthood, with a mean age of sudden death of 41 +/- 15 years, but also occurs in infants and children. While relatively rare, Brugada Syndrome is a major cause of sudden unexplained death syndrome (SUDS) and is the most common cause of sudden death in young men without known underlying cardiac disease. Most patients are asymptomatic and will not experience malignant arrhythmias. This condition occurs much more frequently in people of Asian ancestry, particularly in Japanese and Southeast Asian populations. Brugada syndrome is usually diagnosed by specific electrocardiographic abnormalities and a physical examination. These abnormalities lead to episodes of abnormal electrical activity, which can cause a dangerous kind of arrhythmia in which the lower chambers (ventricles) beat so fast (ventricular tachycardia or ventricular fibrillation) that the heart cannot pump the blood that it needs for the brain to work normally. Arrhythmia episodes can occur suddenly, leading to fainting, or sometimes to cardiac arrest and sudden death. Even for the approximately 20% who can be diagnosed through genetic testing, the cost of the test is about $4,000. In view of the foregoing, there is a need for a reliable and cost-efficient means for diagnosing Brugada
Syndrome.
SUMMARY OF THE INVENTION
The invention addresses this need. 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. The data presented herein indicates that samples obtained from patients diagnosed with Brugada Syndrome exhibited a decreased level of MESP1.
Accordingly, the invention provides a method of diagnosing Brugada Syndrome in a subject. In exemplary embodiments, the method comprises measuring the level of MESP1 protein or nucleic acid, using an MESP1 binding agent. A test sample is provided from the subject, wherein the sample comprises a circulating cell or a bodily fluid, an in vitro reaction is performed, yielding a complex comprising of an MESP1 protein or nucleic acid and an MESP1- specific binding agent. A decrease in the level of the complex (e.g., at least 10%, 20%, 30%, 40%, 50%, or more) compared to a normal control indicates an increased risk of BrS, e.g., a high risk of BrS.
The methods described herein represent a non-invasive (or minimally invasive) test assay. For example, the 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. In another example, the test sample comprises saliva. In another example, the 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. Typically the subject is a human being characterized as comprising a risk of heart disease, e.g. , a previous cardiac event, a family history of heart disease, or other risk factor such as obesity or diabetes. In various embodiments, 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.
In various embodiments, 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,
cardiomyopathy (enlarged heart), a thrombosis, a carotid or coronary artery disease, or any combination thereof. In some embodiments, the subject has at least one grandparent, parent, sibling, or child who has been diagnosed with BrS . In certain embodiments, 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
(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.
In various embodiments, the subject is male. In some embodiments, the subject self- identifies as of Asian descent. In some embodiments, the subject self-identifies as of Japanese descent. In some embodiments, the subject self-identifies as of Southeast Asian (e.g., Bornean, Cambodian, East Timor, Indonesian, Laotian, Malaysian, Burmese, Philippino, Singaporean, Thai, or Vietnamese) descent.
In certain embodiments, 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.
In some embodiments, 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. In various embodiments, 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). For example, a subject has been tested for such a SCN5A abnormality, and the test was negative. In certain embodiments, 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) 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. In some aspects, 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). For example, 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. Exemplary examples of 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
125 labeled with a detectable moiety, e.g. , a fluorescent compound or a radioactive agent (e.g. , I). When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine. The antibody can also be detectably
152
labeled using fluorescence emitting metals such as Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as
diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). 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. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
According to the invention, 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. As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not
significantly bind to the binding agent specific for the target molecule. Preferably, the level of binding to a biomolecule other than the target molecule results in a binding affinity which is at most only 10% or less, only 5% or less only 2% or less or only 1% or less of the affinity to the target molecule, respectively. A preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity. For example, an antibody has a binding affinity in the low micromolar (10"6), nanomolar (10"7-10"9), with high affinity antibodies in the
-9 -12
low nanomolar (10" ) or pico molar (10" ) range for its specific target ligand.
In some aspects, 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). In
embodiments relating to the use of a nucleic acid probe attached to a solid support (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. In some embodiments involving a probe on a solid support, 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. , on a microarray), hybridized to a probe and then amplified, or amplified and then hybridized to a probe. In some example, a strip may be a nucleic acid-probe coated porous or non-porous solid support strip comprising linking a nucleic acid probe to a carrier to prepare a conjugate and immobilizing the conjugate on a porous solid support. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present subject matter. The support material may have any structural configuration so long as the coupled molecule is capable of binding to a binding agent (e.g. , an antibody). Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a plate (or a well within a multiwell plate), sheet, or test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
In certain embodiments, 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. In some cases, the cells, e.g. , white blood cells, are lysed to yield a cell lysate prior to contacting the test sample (cell or cell lysate) with an MESP1 binding agent. In some embodiments, detection is accomplished using an enzyme- linked immunosorbent assay (ELISA) or Western blot format. In other examples, 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. In various embodiments, 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.
In certain embodiments, 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.
Various implementations comprise physically lysing cells, such as by mechanical disruption, liquid homogenization, high frequency sound waves, freeze/thaw cycles and manual grinding. In some embodiments, a detergent is used to lyse cells. Non-limiting examples of detergents include Tween 20, Triton X- 100, and Sodium Dodecyl Sulfate (SDS). Non-limiting examples of assays for determining the level of a protein include Western blot, ELISA, and protein gel electrophoresis. Non-limiting examples of assays for determining the level of an mRNA include Northern blotting, reverse transcription is followed by quantitative polymerase chain reaction (RT-PCR), and reverse transcription followed by quantitative PCR (RT-qPCR).
The term "sample" as used herein refers to a biological sample obtained for the purpose of evaluation in vitro. With regard to the methods disclosed herein, the sample or patient sample preferably may comprise any body fluid. In some embodiments, 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. In some aspects, the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample. In exemplary aspects, the sample comprises blood or a fraction thereof (e.g. , plasma, serum, fraction obtained via leukopheresis). In exemplary aspects, the sample comprises white blood cells obtained from the subject. In exemplary aspects, the sample comprises only white blood cells. In exemplary aspects, the sample is cardiac tissue (e.g. , cardiac muscle tissue). Preferred samples are whole blood, serum, plasma, or urine.
Optionally, 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). Optionally, the method may also include the step of treatment following risk stratification as described above.
Thus, 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. Upon observation of a decreased MESPl protein or an MESPl nucleic acid level, an MESPl elevating medication is administered to the patient. For example, 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.
For purposes herein, 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. In exemplary aspects, the anti-arrhythmic agent is a Singh Vaughan Williams (SVW) Class I, II, III, IV, or V anti-arrhythmic agent. In exemplary aspects, 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. In some aspects, the SVW Class IA is Quinidine, Procainamide, or Disopyramide. In some aspects, the SVW Class IB antiarrhythmic agent is Lidocaine, Phenytoin, Mexiletine, or Tocainide. In some aspects, the SVW Class IC anti-arrhythmic agent is Flecainide, Propafenone, Moricizine, or Encainide. In some aspects, the SVW Class III anti-arrhythmic agent is Dronedarone,
Amiodarone, or Ibutilide. In some aspects, the anti-arrhythmic agent is NAD+ or mitoTEMPO. Also within the invention is a kit comprising an MESPl binding agent and instructions for using the agent for evaluating arrhythmic risk or assessing the severity of heart failure. In some embodiments, 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. In some cases such 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. , to liberate an MESPl protein or an MESPl nucleic acid such as mRNA from the cell to permit measurement of the protein or gene transcript). The agent may be attached to a solid support (e.g. , a test strip). A complex intermediate of MESPl mRNA and test agent may be formed to detect such liberated MESPl nucleic acid. Still another embodiment of the invention is a kit 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. For example, 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. In various embodiments, the written report is electronic. Alternatively or in addition, 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.
In some implementations of the present subject matter, evaluating the presence of Brugada Syndrome, assessing the risk of arrhythmia or SCD, determining prognosis, or assessing Brugada Syndrome severity is performed alone or together with the consideration of other variables, (e.g., as part of an array of testing), to establish or confirm the absence or presence of Brugada Syndrome, or aid the physician in the prognosis, and or the monitoring of treatment. The skilled artisan will appreciate that any such evaluation or assessment made based on the level of a biomarker as determined using in vitro assay. In various embodiments, a patient's 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. In certain embodiments, the sample is a liquid sample, e.g., whole blood, serum, or plasma.
Certain embodiments relate to the use of a method disclosed herein as part of an array of testing. For example, 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.
Aspects of the present subject matter provide methods of treating a subject. In some embodiments, the subject has been diagnosed with Brugada Syndrome according to a method disclosed herein. In some embodiments, 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. For example, 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).
Therapies for Brugada Syndrome and/or reducing the risk of SCD include administration of one or more antiarrhythmic agents. Non-limiting examples of 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. In exemplary aspects, the anti-arrhythmic agent is a Singh Vaughan Williams (SVW) Class I, II, III, IV, or V anti- arrhythmic agent. In exemplary aspects, 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. In some aspects, the SVW Class IA is Quinidine, Procainamide, or Disopyramide. In some aspects, the SVW Class IB antiarrhythmic agent is Lidocaine, Phenytoin, Mexiletine, or Tocainide. In some aspects, the SVW Class IC antiarrhythmic agent is Flecainide, Propafenone, Moricizine, or Encainide. In some aspects, the SVW Class III anti- arrhythmic agent is Dronedarone, Amiodarone, or Ibutilide. In some aspects, the anti- arrhythmic agent is NAD+ or mitoTEMPO.
In certain embodiments, treating Brugada Syndrome and/or reducing the risk of SCD comprises implantation of an automatic implantable cardiac defibrillator (ICD).
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. In some embodiments, the amount of a treatment may be adjusted upon observation of a change in the level of a biomarker. In various embodiments, 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. , over a period of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. Since the accurate diagnosis of a subject leads to determining the appropriate therapy for treating the diagnosed medical condition, disease, or syndrome, the present subject matter also provides related methods of determining need for therapy or prophylaxis of a subject. For example, 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. In various embodiments, 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.
In some embodiments, a method to monitor the efficacy of treatment is provided. In such embodiments, the method comprises determining a level of a biomarker in a test sample of a subject before and after treatment. In such a method, 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. In some embodiments, a first test sample is obtained from the subject to be treated prior to initiation of therapy or part way through a therapy regime. In various embodiments, a test sample is provided to a person performing the assay. Alternatively, in some embodiments, a first test sample is obtained or provided from a subject known not to suffer from a condition being treated. In some embodiments, 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. For example, an increase in the level of MESP1 in the second test sample (e.g. , post-treatment) compared to 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) indicates a degree of effective therapy. Additionally, an increase in the level of full-length SCN5A in the second test sample (e.g. , post-treatment) compared to the level of full-length SCN5A 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. 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.
Aspects of the present subject matter provide a method for diagnosing Brugada
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. In some embodiments, 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. In various embodiments, 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 MESPl protein-encoding mRNA in the test sample. In some embodiments, 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. In certain embodiments, wherein the database contains (a) level values of MESPl protein or MESPl protein-encoding mRNA from (i) subjects who have suffered from SCD, (ii) subjects who are afflicted with Brugada Syndrome but who have not suffered from SCD, (iii) subjects afflicted with Brugada Syndrome for at least about 1, 2, 3, 4, 5, 10, 15, or 20 years without suffering from SCD, and/or (iv) subjects who are not afflicted with Brugada Syndrome, and/or (b) mean or median level values calculated using MESP1 protein or MESP1 protein-encoding mRNA level values from (i) subjects who have suffered from SCD, (ii) subjects who are afflicted with Brugada Syndrome but who have not suffered from SCD, (iii) subjects afflicted with Brugada Syndrome for at least about 1, 2, 3, 4, 5, 10, 15, or 20 years without suffering from SCD, and/or (iv) subjects who are not afflicted with Brugada Syndrome.
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. In various embodiments, the therapy is a test therapy. For example, the subject may be enrolled in a clinical trial.
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 following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. Abbreviations used herein have their conventional meaning within the chemical and biological arts.
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). The term "about" with respect to 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. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. In some embodiments, the term "about" in the context of a numerical value or range may mean +10% of the numerical value or range recited or claimed.
It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, "0.2-5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
The terms "administration" or "administering" refer to the act of providing an agent of the current embodiments or pharmaceutical composition including an agent of the current embodiments to the individual in need of treatment.
The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise.
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. Generally, 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.
The 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. By contrast, the transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.
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
representative of measured levels of markers, wherein said measured levels of markers comprise the levels of a set or panel of markers; and means for reporting said index value. In some variations of the diagnostic test system, the index value is arrhythmia risk score. In some preferred variations, the arrhythmia risk score is computed according to the methods described herein for computing such scores. In some variations, the means for collecting and tracking test results data representing for one or more individuals comprises a data structure or database. In some variations, 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.
The term "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. The skilled artisan will appreciate that 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. Typically, the sample is a liquid sample, e.g., whole blood, serum or plasma.
By "fluoroimmunoassay" is meant an assay that has an agent labeled with a fluorophore. By "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. 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. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid
(mRNA) molecules. cDNA is not naturally occurring.
By "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).
The term "modulate", "modulating" as used herein means regulating or adjusting to a certain degree.
The term, "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. In some aspects, 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.
Relative to a control level, the level that is determined may an increased level. As used herein, the term "increased" with respect to level (e.g., expression level, biological activity 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.
Relative to a control level, the level that is determined may a decreased level. As used herein, the term "decreased" with respect to level (e.g., expression level, biological activity 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.
As used herein, the term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA, e.g. cDNA), and, where appropriate, ribonucleic acid (RNA). 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. Specifically, as used herein, 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.
Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, 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
deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally- occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
By "radioimmunoassay" (RIA) is meant as an in vitro assay used to measure
concentrations of agents using an antibody, by use of labeled antigen (e.g., gamma-radioactive isotopes of iodine).
Receiver-operating characteristics (ROC) 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
Youden's Index). The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups.
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. In each case, 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. 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 (no overlap in the two distributions of results) 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 (identical distributions of results for the two groups) 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). Qualitatively, 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. Such an overall parameter, e.g., is the so-called "total error" or alternatively the "area under the curve=AUC". The most common global measure is the area under the ROC plot. By convention, this area is always >0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area=1.0).
"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. In some
embodiments, 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.
As used herein, the term "salt" refers to acid or base salts of the agents used herein. Illustrative but non-limiting examples of 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 (e.g. , Brugada Syndrome) 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. Alternatively, 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.
The term "stabilizing agent" refers to a small molecule, an antibody (or fragment thereof), or a binding molecule that yields a complex that does not readily become inactive or denature, for example reversible [e.g. , formaldehyde, SPDP (succinimidyl 3-(3-pyridyldithio propionate)] and non-reversible cross-linkers.
The term "subject" as used herein includes all members of the animal kingdom prone to suffering from the indicated disorder. For example, the term "subject" as used herein includes all members of the animal kingdom that suffer from or may suffer from the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human. The methods are also applicable to companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs, and other domesticated and wild animals.
By "substantially pure" is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, 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. With respect to a cell type, 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. For example, 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. In a particular example, an isolated CD4 positive cell population is a population of CD4 positive cells that is substantially separated or purified away from other blood cells, such as CD8 positive cells. In one example, an isolated CD4 positive T-cell population is at least 95% pure, such as at least 99% pure. In another particular example, an isolated B-cell population is a population of B-cells that is substantially separated or purified away from other blood cells, such as T-cells. In one example, an isolated B- cell population is at least 95% pure, such as at least 99% pure. An enriched population of white blood cells (e.g., buffy coat fraction) is a population that have been separated from red blood cells, e.g., by density gradient.
As used herein, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this disclosure, 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.
Insofar as the methods of the present disclosure are directed to 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.
As used herein, "assaying" means using an analytic procedure to qualitatively assess or quantitatively measure the presence or amount or the functional activity of a target entity. For example, assaying the level of a compound (such as a protein or an mRNA molecule) means using an analytic procedure (such as an in vitro procedure) to qualitatively assess or
quantitatively measure the presence or amount of the compound.
In the descriptions above and in the claims, 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. For example, the phrases "at least one of A and Β;" "one or more of A and Β;" 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. For example, 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." In addition, 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.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are hereby incorporated by reference. DETAILED DESCRIPTION OF THE INVENTION
Brugada syndrome (BrS)
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
(electrocardiogram), with no structural abnormalities. This defect can lead to episodes of abnormal electrical activity, which can cause a dangerous kind of arrhythmia in which the lower chambers (ventricles) beat so fast (ventricular tachycardia or ventricular fibrillation) that the heart cannot pump the blood it needs for the brain to work normally. Arrhythmia episodes can occur suddenly, leading to fainting, or sometimes to cardiac arrest and sudden death. The exact prevalence of BrS is unknown, although it is estimated to affect 5 in 10,000 people worldwide and approximately 155,000 people in the United States. While relatively rare, BrS is a major cause of sudden unexplained death syndrome (SUDS) and is the most common cause of sudden death in young men without known underlying cardiac disease. This condition occurs much more frequently in people of Asian ancestry, particularly in Japanese and Southeast Asian populations. Although BrS affects both men and women, the condition appears to be 8 to 10 times more common in men. Researchers suspect that testosterone, a sex hormone present at much higher levels in men, may be responsible for this difference.
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.
To manage BrS, doctors frequently recommend either an electrophysiology (EP) study to monitor the condition or a special device that is implanted near the shoulder called an
implantable cardioverter defibrillator (ICD) that can prevent an arrhythmia attack.
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.
Arrhythmias
The invention further comprises a subject's risk for arrhythmia. As used herein, the term "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.
Bradycardia
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. Alternatively, the bradycardia is caused by a slowed electrical conduction in the heart.
Tachycardia
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. Alternatively, 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.
Sudden Cardiac Death
Brugada Syndrome can lead to sudden cardiac death (SCD). 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).
When this occurs, the heart is unable to pump blood and death will occur within minutes, if left untreated.
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.
Human MESP1 amino acid sequence
1 maqplcppls eswmlsaawg ptrrpppsdk dcgrslvssp dswgstpads pvasparpgt 61 lrdprapsvg rrgarssrlg sgqrqsaser eklrmrtlar alhelrrflp psvapagqsl 121 tkietlrlai ryighlsavl glseeslqrr crqrgdagsp rgcplcpddc paqmqtrtqa 181 egqgqgrglg lvsavragas wgsppacpga raapeprdpp alfaeaacpe gqamepspps 241 pllpgdvlal letwmplspl ewlpeepk
(SEQ ID NO: 1) GenBank Accession NP_061140.1 (GI: 14149724), incorporated herein by reference.
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.
Human MESPl nucleotide sequence. The start and stop codons of the coding sequence are bold and underlined.
1 tggaaggggc cacttcacac ctcgggctcg gcataaagcg gccgccggcc gccggccccc
61 agacgcgccg ccgctgccat ggcccagccc ctgtgcccgc cgctctccga gtcctggatg
121 ctctctgcgg cctggggccc aactcggcgg ccgccgccct ccgacaagga ctgcggccgc
181 tccctcgtct cgtccccaga ctcatggggc agcaccccag ccgacagccc cgtggcgagc
241 cccgcgcggc caggcaccct ccgggacccc cgcgccccct ccgtaggtag gcgcggcgcg
301 cgcagcagcc gcctgggcag cgggcagagg cagagcgcca gtgagcggga gaaactgcgc
361 atgcgcacgc tggcccgcgc cctgcacgag ctgcgccgct ttctaccgcc gtccgtggcg
421 cccgcgggcc agagcctgac caagatcgag acgctgcgcc tggctatccg ctatatcggc
481 cacctgtcgg ccgtgctagg cctcagcgag gagagtctcc agcgccggtg ccggcagcgc
541 ggtgacgcgg ggtcccctcg gggctgcccg ctgtgccccg acgactgccc cgcgcagatg
601 cagacacgga cgcaggctga ggggcagggg caggggcgcg ggctgggcct ggtatccgcc
661 gtccgcgccg gggcgtcctg gggatccccg cctgcctgcc ccggagcccg agctgcaccc
721 gagccgcgcg acccgcctgc gctgttcgcc gaggcggcgt gccctgaagg gcaggcgatg
781 gagccaagcc caccgtcccc gctccttccg ggcgacgtgc tggctctgtt ggagacctgg
841 atgcccctct cgcctctgga gtggctgcct gaggagccca agtgacaagg gacaactgac
901 gccgtctctg tgagcaccga ggctttttgg cctcagcacc ttcgaagtgg ttccttggca
961 gactgccttt cctggaagag ggcacgggcg atcccgacgg gggcattcct gcgggtgaga
1021 gccgtcccca ccgcggcggc ccttctcagc ccctccctcc atggagggac ccatagggct
1081 agacactttg aggcaagcag gaggctctgc ctaatgtgaa tttatttatt tgtgaataaa
1141 ctgtactggt gtcagttggc aaaaaaaaaa aaaaaaaaaa aaaa
(SEQ ID NO: 2) GenBank Accession NMJ318670.3 (GI: 219842275), incorporated herein by reference.
Exemplary regions for MESPl include bases 565-579 (encodes CPLCP), 622-633 (encodes 2 X 2 AA tandem repeats of G-Q), and 802-1162. SCN5A (Sodium Channel, Voltage Gated, Type V alpha Protein)
Cardiac voltage-gated Na+ (Nav) channels consist of a heteromeric assembly of pore- forming a subunit and auxiliary β subunits that modulate channel functions. Nav 1.5 (SCN5A) is the major Nav a subunit expressed in the mammalian myocardium, whereas multiple Nav β 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
myocardium. Abnormal cardiac Na+ channel function has been associated in hereditary cardiac diseases such as long QT syndrome (LQTS), Brugada syndrome, and progressive cardiac conduction defect, as well as acquired cardiac conditions including myocardial ischemia and heart failure.
The method of identifying a subject at risk for arrhythmias or heart failure comprises the step of determining a level of a full length transcript of SCN5A gene or of a splice variant of the SCN5A gene. A decreased level of the full length transcript of the SCN5A gene indicates an increased risk for arrhythmia or heart failure. In exemplary aspects, a level of a splice variant of the SCN5A gene is determined, and an increased level of the splice variant indicates an increased risk for arrhythmia or heart failure. In specific aspects, the splice variant of the SCN5A gene is a splice variant produced from alternative splicing within Exon 28 of the SCN5A gene. In specific aspects, 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.
SCN5A splice variant Exon 28 B (E28B)
ggagccctcc tagtgagtat gaagtgatat ctcactgagg ttttggtttg caaaagcaaa 60 tgactgatga ctaacgatgc aggacatctt tccatgtgca tgttggtcat ttatatatct 120 tccttggaga aatctctatt cagatcctta gctcattttt aattgggtta tttctcttct 180 tcttgttgag ttgtaagagt tctttacata ttctggatca cagtctctta tcagatatat 240 gatttaaaaa tattttctcc tagtctgtga gttttttcat ttcctagtgg tgtccattaa 300 agcacaaaag ttttacatgt t 321
(SEQ ID NO: 3)
SCN5A splice variant Exon 28 C (E28C)
gaactgcaca atgaccagca ggaggggaga agagagtagg aaaaaggagg gaaggacaga 60 catcaagtgc cagatgttgt ctgaactaat cgagcacttc tcaccaaact tcatgtataa 120 ataaaataca tatttttaaa acaaaccaat aaatggctta catg 164 (SEQ ID NO: 4)
SCN5A splice variant Exon 28 D (E28D)
ggcactgtgc tctcggacat catccagaag tacttcttct ccccgacgct cttccgagtc 60 atccgcctgg cccgaatagg ccgcatcctc agactgatcc gaggggccaa gggg 114
(SEQ ID NO: 5)
E28B SCN5A Splice Variant Complete Nucleotide Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
1 agacggcggc ggcgcccgta ggatgcaggg atcgctcccc cggggccgct gagcctgcgc 61 ccagtgcccc gagccccgcg ccgagccgag tccgcgccaa gcagcagccg cccaccccgg 121 ggcccggccg ggggaccagc agcttcccca caggcaacgt gaggagagcc tgtgcccaga 181 agcaggatga gaagatggca aacttcctat tacctcgggg caccagcagc ttccgcaggt 241 tcacacggga gtccctggca gccatcgaga agcgcatggc agagaagcaa gcccgcggct 301 caaccacctt gcaggagagc cgagaggggc tgcccgagga ggaggctccc cggccccagc 361 tggacctgca ggcctccaaa aagctgccag atctctatgg caatccaccc caagagctca 421 tcggagagcc cctggaggac ctggacccct tctatagcac ccaaaagact ttcatcgtac 481 tgaataaagg caagaccatc ttccggttca gtgccaccaa cgccttgtat gtcctcagtc 541 ccttccaccc catccggaga gcggctgtga agattctggt tcactcgctc ttcaacatgc 601 tcatcatgtg caccatcctc accaactgcg tgttcatggc ccagcacgac cctccaccct 661 ggaccaagta tgtcgagtac accttcaccg ccatttacac ctttgagtct ctggtcaaga 721 ttctggctcg aggcttctgc ctgcacgcgt tcactttcct tcgggaccca tggaactggc 781 tggactttag tgtgattatc atggcataca caactgaatt tgtggacctg ggcaatgtct 841 cagccttacg caccttccga gtcctccggg ccctgaaaac tatatcagtc atttcagggc 901 tgaagaccat cgtgggggcc ctgatccagt ctgtgaagaa gctggctgat gtgatggtcc 961 tcacagtctt ctgcctcagc gtctttgccc tcatcggcct gcagctcttc atgggcaacc 1021 taaggcacaa gtgcgtgcgc aacttcacag cgctcaacgg caccaacggc tccgtggagg 1081 ccgacggctt ggtctgggaa tccctggacc tttacctcag tgatccagaa aattacctgc 1141 tcaagaacgg cacctctgat gtgttactgt gtgggaacag ctctgacgct gggacatgtc 1201 cggagggcta ccggtgccta aaggcaggcg agaaccccga ccacggctac accagcttcg 1261 attcctttgc ctgggccttt cttgcactct tccgcctgat gacgcaggac tgctgggagc
1321 gcctctatca gcagaccctc aggtccgcag ggaagatcta catgatcttc ttcatgcttg
1381 tcatcttcct ggggtccttc tacctggtga acctgatcct ggccgtggtc gcaatggcct
1441 atgaggagca aaaccaagcc accatcgctg agaccgagga gaaggaaaag cgcttccagg
1501 aggccatgga aatgctcaag aaagaacacg aggccctcac catcaggggt gtggataccg
1561 tgtcccgtag ctccttggag atgtcccctt tggccccagt aaacagccat gagagaagaa
1621 gcaagaggag aaaacggatg tcttcaggaa ctgaggagtg tggggaggac aggctcccca
1681 agtctgactc agaagatggt cccagagcaa tgaatcatct cagcctcacc cgtggcctca
1741 gcaggacttc tatgaagcca cgttccagcc gcgggagcat tttcaccttt cgcaggcgag
1801 acctgggttc tgaagcagat tttgcagatg atgaaaacag cacagcgggg gagagcgaga
1861 gccaccacac atcactgctg gtgccctggc ccctgcgccg gaccagtgcc cagggacagc
1921 ccagtcccgg aacctcggct cctggccacg ccctccatgg caaaaagaac agcactgtgg
1981 actgcaatgg ggtggtctca ttactggggg caggcgaccc agaggccaca tccccaggaa
2041 gccacctcct ccgccctgtg atgctagagc acccgccaga cacgaccacg ccatcggagg
2101 agccaggcgg gccccagatg ctgacctccc aggctccgtg tgtagatggc ttcgaggagc
2161 caggagcacg gcagcgggcc ctcagcgcag tcagcgtcct caccagcgca ctggaagagt
2221 tagaggagtc tcgccacaag tgtccaccat gctggaaccg tctcgcccag cgctacctga
2281 tctgggagtg ctgcccgctg tggatgtcca tcaagcaggg agtgaagttg gtggtcatgg
2341 acccgtttac tgacctcacc atcactatgt gcatcgtact caacacactc ttcatggcgc
2401 tggagcacta caacatgaca agtgaattcg aggagatgct gcaggtcgga aacctggtct
2461 tcacagggat tttcacagca gagatgacct tcaagatcat tgccctcgac ccctactact
2521 acttccaaca gggctggaac atcttcgaca gcatcatcgt catccttagc ctcatggagc
2581 tgggcctgtc ccgcatgagc aacttgtcgg tgctgcgctc cttccgcctg ctgcgggtct
2641 tcaagctggc caaatcatgg cccaccctga acacactcat caagatcatc gggaactcag
2701 tgggggcact ggggaacctg acactggtgc tagccatcat cgtgttcatc tttgctgtgg
2761 tgggcatgca gctctttggc aagaactact cggagctgag ggacagcgac tcaggcctgc
2821 tgcctcgctg gcacatgatg gacttctttc atgccttcct catcatcttc cgcatcctct
2881 gtggagagtg gatcgagacc atgtgggact gcatggaggt gtcggggcag tcattatgcc
2941 tgctggtctt cttgcttgtt atggtcattg gcaaccttgt ggtcctgaat ctcttcctgg
3001 ccttgctgct cagctccttc agtgcagaca acctcacagc ccctgatgag gacagagaga
3061 tgaacaacct ccagctggcc ctggcccgca tccagagggg cctgcgcttt gtcaagcgga
3121 ccacctggga tttctgctgt ggtctcctgc ggcagcggcc tcagaagccc gcagcccttg
3181 ccgcccaggg ccagctgccc agctgcattg ccacccccta ctccccgcca cccccagaga
3241 cggagaaggt gcctcccacc cgcaaggaaa cacggtttga ggaaggcgag caaccaggcc
3301 agggcacccc cggggatcca gagcccgtgt gtgtgcccat cgctgtggcc gagtcagaca
3361 cagatgacca agaagaagat gaggagaaca gcctgggcac ggaggaggag tccagcaagc
3421 agcaggaatc ccagcctgtg tccggtggcc cagaggcccc tccggattcc aggacctgga
3481 gccaggtgtc agcgactgcc tcctctgagg ccgaggccag tgcatctcag gccgactggc
3541 ggcagcagtg gaaagcggaa ccccaggccc cagggtgcgg tgagacccca gaggacagtt
3601 gctccgaggg cagcacagca gacatgacca acaccgctga gctcctggag cagatccctg
3661 acctcggcca ggatgtcaag gacccagagg actgcttcac tgaaggctgt gtccggcgct
3721 gtccctgctg tgcggtggac accacacagg ccccagggaa ggtctggtgg cggttgcgca
3781 agacctgcta ccacatcgtg gagcacagct ggttcgagac attcatcatc ttcatgatcc
3841 tactcagcag tggagcgctg gccttcgagg acatctacct agaggagcgg aagaccatca
3901 aggttctgct tgagtatgcc gacaagatgt tcacatatgt cttcgtgctg gagatgctgc
3961 tcaagtgggt ggcctacggc ttcaagaagt acttcaccaa tgcctggtgc tggctcgact
4021 tcctcatcgt agacgtctct ctggtcagcc tggtggccaa caccctgggc tttgccgaga
4081 tgggccccat caagtcactg cggacgctgc gtgcactccg tcctctgaga gctctgtcac
4141 gatttgaggg catgagggtg gtggtcaatg ccctggtggg cgccatcccg tccatcatga
4201 acgtcctcct cgtctgcctc atcttctggc tcatcttcag catcatgggc gtgaacctct
4261 ttgcggggaa gtttgggagg tgcatcaacc agacagaggg agacttgcct ttgaactaca 4321 ccatcgtgaa caacaagagc cagtgtgagt ccttgaactt gaccggagaa ttgtactgga 4381 ccaaggtgaa agtcaacttt gacaacgtgg gggccgggta cctggccctt ctgcaggtgg 4441 caacatttaa aggctggatg gacattatgt atgcagctgt ggactccagg gggtatgaag 4501 agcagcctca gtgggaatac aacctctaca tgtacatcta ttttgtcatt ttcatcatct 4561 ttgggtcttt cttcaccctg aacctcttta ttggtgtcat cattgacaac ttcaaccaac 4621 agaagaaaaa gttagggggc caggacatct tcatgacaga ggagcagaag aagtactaca 4681 atgccatgaa gaagctgggc tccaagaagc cccagaagcc catcccacgg cccctgaaca 4741 agtaccaggg cttcatattc gacattgtga ccaagcaggc ctttgacgtc accatcatgt 4801 ttctgatctg cttgaatatg gtgaccatga tggtggagac agatgaccaa agtcctgaga 4861 aaatcaacat cttggccaag atcaacctgc tctttgtggc catcttcaca ggcgagtgta 4921 ttgtcaagct ggctgccctg cgccactact acttcaccaa cagctggaat atcttcgact 4981 tcgtggttgt catcctctcc atcgtgggag ccctcctagt gagtatgaag tgatatctca 5041 ctgaggtttt ggtttgcaaa agcaaatgac tgatgactaa cgatgcagga catctttcca 5101 tgtgcatgtt ggtcatttat atatcttcct tggagaaatc tctattcaga tccttagctc 5161 atttttaatt gggttatttc tcttcttctt gttgagttgt aagagttctt tacatattct 5221 ggatcacagt ctcttatcag atatatgatt taaaaatatt ttctcctagt ctgtgagttt 5281 tttcatttcc tagtggtgtc cattaaagca caaaagtttt acatgtt
(SEQ ID NO: 6)
E28B SCN5A Splice Variant Complete Amino Acid Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
1 manfllprgt ssfrrftres laaiekrmae kqargsttlq esreglpeee aprpqldlqa 61 skklpdlygn ppqeligepl edldpfystq ktfivlnkgk tifrfsatna lyvlspfhpi 121 rraavkilvh slfnmlimct iltncvfmaq hdpppwtkyv eytftaiytf eslvkilarg 181 fclhaftflr dpwnwldfsv iimayttefv dlgnvsalrt frvlralkti svisglktiv 241 galiqsvkkl advmvltvfc lsvfaliglq lfmgnlrhkc vrnftalngt ngsveadglv 301 wesldlylsd penyllkngt sdvllcgnss dagtcpegyr clkagenpdh gytsfdsfaw 361 aflalfrlmt qdcwerlyqq tlrsagkiym iffmlviflg sfylvnlila vamayeeqn 421 qatiaeteek ekrfqeamem lkkehealti rgvdtvsrss lemsplapvn sherrskrrk 481 rmssgteecg edrlpksdse dgpramnhls ltrglsrtsm kprssrgsif tfrrrdlgse 541 adfaddenst ageseshhts llvpwplrrt saqgqpspgt sapghalhgk knstvdcngv 601 vsllgagdpe atspgshllr pvmlehppdt ttpseepggp qmltsqapcv dgfeepgarq 661 ralsavsvlt saleeleesr hkcppcwnrl aqryliwecc plwmsikqgv klvvmdpftd 721 ltitmcivln tlfmalehyn mtsefeemlq vgnlvftgif taemtfkiia ldpyyyfqqg 781 wnifdsiivi lslmelglsr msnlsvlrsf rllrvfklak swptlntlik iignsvgalg 841 nltlvlaiiv fifavvgmql fgknyselrd sdsgllprwh mmdffhafli ifrilcgewi 901 etmwdcmevs gqslcllvfl lvmvignlvv lnlflallls sfsadnltap dedremnnlq 961 lalariqrgl rfvkrttwdf ccgllrqrpq kpaalaaqgq lpsciatpys ppppetekvp 1021 ptrketrfee geqpgqgtpg dpepvcvpia vaesdtddqe edeenslgte eesskqqesq 1081 pvsggpeapp dsrtwsqvsa tasseaeasa sqadwrqqwk aepqapgcge tpedscsegs 1141 tadmtntael leqipdlgqd vkdpedcfte gcvrrcpcca vdttqapgkv wwrlrktcyh 1201 ivehswfetf iifmillssg alafediyle erktikvlle yadkmftyvf vlemllkwva 1261 ygfkkyftna wcwldflivd vslvslvant Igfaemgpik slrtlralrp Iralsrfegm 1321 rvvvnalvga ipsimnvllv clifwlifsi mgvnlfagkf grcinqtegd lplnytivnn 1381 ksqceslnlt gelywtkvkv nfdnvgagyl allqvatfkg wmdimyaavd srgyeeqpqw 1441 eynlymyiyf vifiifgsff tlnlfigvii dnfnqqkkkl ggqdifmtee qkkyynamkk 1501 lgskkpqkpi prplnkyqgf ifdivtkqaf dvtimflicl nmvtmmvetd dqspekinil 1561 akinllfvai ftgecivkla alrhyyftns wnifdfvvvi lsivgallvs mk
(SEQ ID NO: 7) E28C SCN5A Splice Variant Complete Nucleotide Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
1 agacggcggc ggcgcccgta ggatgcaggg atcgctcccc cggggccgct gagcctgcgc 61 ccagtgcccc gagccccgcg ccgagccgag tccgcgccaa gcagcagccg cccaccccgg 121 ggcccggccg ggggaccagc agcttcccca caggcaacgt gaggagagcc tgtgcccaga 181 agcaggatga gaagatggca aacttcctat tacctcgggg caccagcagc ttccgcaggt 241 tcacacggga gtccctggca gccatcgaga agcgcatggc agagaagcaa gcccgcggct 301 caaccacctt gcaggagagc cgagaggggc tgcccgagga ggaggctccc cggccccagc 361 tggacctgca ggcctccaaa aagctgccag atctctatgg caatccaccc caagagctca 421 tcggagagcc cctggaggac ctggacccct tctatagcac ccaaaagact ttcatcgtac 481 tgaataaagg caagaccatc ttccggttca gtgccaccaa cgccttgtat gtcctcagtc 541 ccttccaccc catccggaga gcggctgtga agattctggt tcactcgctc ttcaacatgc 601 tcatcatgtg caccatcctc accaactgcg tgttcatggc ccagcacgac cctccaccct 661 ggaccaagta tgtcgagtac accttcaccg ccatttacac ctttgagtct ctggtcaaga 721 ttctggctcg aggcttctgc ctgcacgcgt tcactttcct tcgggaccca tggaactggc 781 tggactttag tgtgattatc atggcataca caactgaatt tgtggacctg ggcaatgtct 841 cagccttacg caccttccga gtcctccggg ccctgaaaac tatatcagtc atttcagggc 901 tgaagaccat cgtgggggcc ctgatccagt ctgtgaagaa gctggctgat gtgatggtcc 961 tcacagtctt ctgcctcagc gtctttgccc tcatcggcct gcagctcttc atgggcaacc 1021 taaggcacaa gtgcgtgcgc aacttcacag cgctcaacgg caccaacggc tccgtggagg 1081 ccgacggctt ggtctgggaa tccctggacc tttacctcag tgatccagaa aattacctgc 1141 tcaagaacgg cacctctgat gtgttactgt gtgggaacag ctctgacgct gggacatgtc 1201 cggagggcta ccggtgccta aaggcaggcg agaaccccga ccacggctac accagcttcg 1261 attcctttgc ctgggccttt cttgcactct tccgcctgat gacgcaggac tgctgggagc 1321 gcctctatca gcagaccctc aggtccgcag ggaagatcta catgatcttc ttcatgcttg 1381 tcatcttcct ggggtccttc tacctggtga acctgatcct ggccgtggtc gcaatggcct 1441 atgaggagca aaaccaagcc accatcgctg agaccgagga gaaggaaaag cgcttccagg 1501 aggccatgga aatgctcaag aaagaacacg aggccctcac catcaggggt gtggataccg 1561 tgtcccgtag ctccttggag atgtcccctt tggccccagt aaacagccat gagagaagaa 1621 gcaagaggag aaaacggatg tcttcaggaa ctgaggagtg tggggaggac aggctcccca 1681 agtctgactc agaagatggt cccagagcaa tgaatcatct cagcctcacc cgtggcctca 1741 gcaggacttc tatgaagcca cgttccagcc gcgggagcat tttcaccttt cgcaggcgag 1801 acctgggttc tgaagcagat tttgcagatg atgaaaacag cacagcgggg gagagcgaga 1861 gccaccacac atcactgctg gtgccctggc ccctgcgccg gaccagtgcc cagggacagc 1921 ccagtcccgg aacctcggct cctggccacg ccctccatgg caaaaagaac agcactgtgg 1981 actgcaatgg ggtggtctca ttactggggg caggcgaccc agaggccaca tccccaggaa 2041 gccacctcct ccgccctgtg atgctagagc acccgccaga cacgaccacg ccatcggagg 2101 agccaggcgg gccccagatg ctgacctccc aggctccgtg tgtagatggc ttcgaggagc 2161 caggagcacg gcagcgggcc ctcagcgcag tcagcgtcct caccagcgca ctggaagagt 2221 tagaggagtc tcgccacaag tgtccaccat gctggaaccg tctcgcccag cgctacctga 2281 tctgggagtg ctgcccgctg tggatgtcca tcaagcaggg agtgaagttg gtggtcatgg 2341 acccgtttac tgacctcacc atcactatgt gcatcgtact caacacactc ttcatggcgc 2401 tggagcacta caacatgaca agtgaattcg aggagatgct gcaggtcgga aacctggtct 2461 tcacagggat tttcacagca gagatgacct tcaagatcat tgccctcgac ccctactact 2521 acttccaaca gggctggaac atcttcgaca gcatcatcgt catccttagc ctcatggagc 2581 tgggcctgtc ccgcatgagc aacttgtcgg tgctgcgctc cttccgcctg ctgcgggtct 2641 tcaagctggc caaatcatgg cccaccctga acacactcat caagatcatc gggaactcag 2701 tgggggcact ggggaacctg acactggtgc tagccatcat cgtgttcatc tttgctgtgg 2761 tgggcatgca gctctttggc aagaactact cggagctgag ggacagcgac tcaggcctgc 2821 tgcctcgctg gcacatgatg gacttctttc atgccttcct catcatcttc cgcatcctct 2881 gtggagagtg gatcgagacc atgtgggact gcatggaggt gtcggggcag tcattatgcc 2941 tgctggtctt cttgcttgtt atggtcattg gcaaccttgt ggtcctgaat ctcttcctgg 3001 ccttgctgct cagctccttc agtgcagaca acctcacagc ccctgatgag gacagagaga 3061 tgaacaacct ccagctggcc ctggcccgca tccagagggg cctgcgcttt gtcaagcgga 3121 ccacctggga tttctgctgt ggtctcctgc ggcagcggcc tcagaagccc gcagcccttg 3181 ccgcccaggg ccagctgccc agctgcattg ccacccccta ctccccgcca cccccagaga 3241 cggagaaggt gcctcccacc cgcaaggaaa cacggtttga ggaaggcgag caaccaggcc 3301 agggcacccc cggggatcca gagcccgtgt gtgtgcccat cgctgtggcc gagtcagaca 3361 cagatgacca agaagaagat gaggagaaca gcctgggcac ggaggaggag tccagcaagc 3421 agcaggaatc ccagcctgtg tccggtggcc cagaggcccc tccggattcc aggacctgga 3481 gccaggtgtc agcgactgcc tcctctgagg ccgaggccag tgcatctcag gccgactggc 3541 ggcagcagtg gaaagcggaa ccccaggccc cagggtgcgg tgagacccca gaggacagtt 3601 gctccgaggg cagcacagca gacatgacca acaccgctga gctcctggag cagatccctg 3661 acctcggcca ggatgtcaag gacccagagg actgcttcac tgaaggctgt gtccggcgct 3721 gtccctgctg tgcggtggac accacacagg ccccagggaa ggtctggtgg cggttgcgca 3781 agacctgcta ccacatcgtg gagcacagct ggttcgagac attcatcatc ttcatgatcc 3841 tactcagcag tggagcgctg gccttcgagg acatctacct agaggagcgg aagaccatca 3901 aggttctgct tgagtatgcc gacaagatgt tcacatatgt cttcgtgctg gagatgctgc 3961 tcaagtgggt ggcctacggc ttcaagaagt acttcaccaa tgcctggtgc tggctcgact 4021 tcctcatcgt agacgtctct ctggtcagcc tggtggccaa caccctgggc tttgccgaga 4081 tgggccccat caagtcactg cggacgctgc gtgcactccg tcctctgaga gctctgtcac 4141 gatttgaggg catgagggtg gtggtcaatg ccctggtggg cgccatcccg tccatcatga 4201 acgtcctcct cgtctgcctc atcttctggc tcatcttcag catcatgggc gtgaacctct 4261 ttgcggggaa gtttgggagg tgcatcaacc agacagaggg agacttgcct ttgaactaca 4321 ccatcgtgaa caacaagagc cagtgtgagt ccttgaactt gaccggagaa ttgtactgga 4381 ccaaggtgaa agtcaacttt gacaacgtgg gggccgggta cctggccctt ctgcaggtgg 4441 caacatttaa aggctggatg gacattatgt atgcagctgt ggactccagg gggtatgaag 4501 agcagcctca gtgggaatac aacctctaca tgtacatcta ttttgtcatt ttcatcatct 4561 ttgggtcttt cttcaccctg aacctcttta ttggtgtcat cattgacaac ttcaaccaac 4621 agaagaaaaa gttagggggc caggacatct tcatgacaga ggagcagaag aagtactaca 4681 atgccatgaa gaagctgggc tccaagaagc cccagaagcc catcccacgg cccctgaaca 4741 agtaccaggg cttcatattc gacattgtga ccaagcaggc ctttgacgtc accatcatgt 4801 ttctgatctg cttgaatatg gtgaccatga tggtggagac agatgaccaa agtcctgaga 4861 aaatcaacat cttggccaag atcaacctgc tctttgtggc catcttcaca ggcgagtgta 4921 ttgtcaagct ggctgccctg cgccactact acttcaccaa cagctggaat atcttcgact 4981 tcgtggttgt catcctctcc atcgtggaac tgcacaatga ccagcaggag gggagaagag 5041 agtaggaaaa aggagggaag gacagacatc aagtgccaga tgttgtctga actaatcgag 5101 cacttctcac caaacttcat gtataaataa aatacatatt tttaaaacaa accaataaat 5161 ggcttacatg
(SEQ ID NO: 8)
E28C SCN5A Splice Variant Complete Amino Acid Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
1 manfllprgt ssfrrftres laaiekrmae kqargsttlq esreglpeee aprpqldlqa 61 skklpdlygn ppqeligepl edldpfystq ktfivlnkgk tifrfsatna lyvlspfhpi 121 rraavkilvh slfnmlimct iltncvfmaq hdpppwtkyv eytftaiytf eslvkilarg 181 fclhaftflr dpwnwldfsv iimayttefv dlgnvsalrt frvlralkti svisglktiv 241 galiqsvkkl advmvltvfc lsvfaliglq lfmgnlrhkc vrnftalngt ngsveadglv 301 wesldlylsd penyllkngt sdvllcgnss dagtcpegyr clkagenpdh gytsfdsfaw 361 aflalfrlmt qdcwerlyqq tlrsagkiym iffmlviflg sfylvnlila vvamayeeqn 421 qatiaeteek ekrfqeamem lkkehealti rgvdtvsrss lemsplapvn sherrskrrk 481 rmssgteecg edrlpksdse dgpramnhls ltrglsrtsm kprssrgsif tfrrrdlgse 541 adfaddenst ageseshhts llvpwplrrt saqgqpspgt sapghalhgk knstvdcngv 601 vsllgagdpe atspgshllr pvmlehppdt ttpseepggp qmltsqapcv dgfeepgarq 661 ralsavsvlt saleeleesr hkcppcwnrl aqryliwecc plwmsikqgv klvvmdpftd 721 ltitmcivln tlfmalehyn mtsefeemlq vgnlvftgif taemtfkiia ldpyyyfqqg 781 wnifdsiivi lslmelglsr msnlsvlrsf rllrvfklak swptlntlik iignsvgalg 841 nltlvlaiiv fifavvgmql fgknyselrd sdsgllprwh mmdffhafli ifrilcgewi 901 etmwdcmevs gqslcllvf1 lvmvignlvv lnlflallls sfsadnltap dedremnnlq 961 lalariqrgl rf krttwdf ccgllrqrpq kpaalaaqgq lpsciatpys ppppetekvp 1021 ptrketrfee geqpgqgtpg dpepvcvpia vaesdtddqe edeenslgte eesskqqesq 1081 pvsggpeapp dsrtwsqvsa tasseaeasa sqadwrqqwk aepqapgcge tpedscsegs 1141 tadmtntael leqipdlgqd vkdpedcfte gcvrrcpcca vdttqapgkv wwrlrktcyh 1201 ivehswfetf iifmillssg alafediyle erktikvlle yadkmftyvf vlemllkwva 1261 ygfkkyftna wcwldflivd vslvslvant Igfaemgpik slrtlralrp Iralsrfegm 1321 rvvvnalvga ipsimnvllv clifwlifsi mgvnlfagkf grcinqtegd lplnytivnn 1381 ksqceslnlt gelywtkvkv nfdnvgagyl allqvatfkg wmdimyaavd srgyeeqpqw 1441 eynlymyiyf vifiifgsff tlnlfigvii dnfnqqkkkl ggqdifmtee qkkyynamkk 1501 lgskkpqkpi prplnkyqgf ifdivtkqaf dvtimflicl nmvtmmvetd dqspekinil 1561 akinllf ai ftgecivkla alrhyyftns wnifdfvvvi lsivelhndq qegrre
(SEQ ID NO: 9)
E28D SCN5A Splice Variant Complete Nucleotide Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
1 agacggcggc ggcgcccgta ggatgcaggg atcgctcccc cggggccgct gagcctgcgc 61 ccagtgcccc gagccccgcg ccgagccgag tccgcgccaa gcagcagccg cccaccccgg 121 ggcccggccg ggggaccagc agcttcccca caggcaacgt gaggagagcc tgtgcccaga 181 agcaggatga gaagatggca aacttcctat tacctcgggg caccagcagc ttccgcaggt 241 tcacacggga gtccctggca gccatcgaga agcgcatggc agagaagcaa gcccgcggct 301 caaccacctt gcaggagagc cgagaggggc tgcccgagga ggaggctccc cggccccagc 361 tggacctgca ggcctccaaa aagctgccag atctctatgg caatccaccc caagagctca 421 tcggagagcc cctggaggac ctggacccct tctatagcac ccaaaagact ttcatcgtac 481 tgaataaagg caagaccatc ttccggttca gtgccaccaa cgccttgtat gtcctcagtc 541 ccttccaccc catccggaga gcggctgtga agattctggt tcactcgctc ttcaacatgc 601 tcatcatgtg caccatcctc accaactgcg tgttcatggc ccagcacgac cctccaccct 661 ggaccaagta tgtcgagtac accttcaccg ccatttacac ctttgagtct ctggtcaaga 721 ttctggctcg aggcttctgc ctgcacgcgt tcactttcct tcgggaccca tggaactggc 781 tggactttag tgtgattatc atggcataca caactgaatt tgtggacctg ggcaatgtct 841 cagccttacg caccttccga gtcctccggg ccctgaaaac tatatcagtc atttcagggc 901 tgaagaccat cgtgggggcc ctgatccagt ctgtgaagaa gctggctgat gtgatggtcc 961 tcacagtctt ctgcctcagc gtctttgccc tcatcggcct gcagctcttc atgggcaacc 1021 taaggcacaa gtgcgtgcgc aacttcacag cgctcaacgg caccaacggc tccgtggagg 1081 ccgacggctt ggtctgggaa tccctggacc tttacctcag tgatccagaa aattacctgc 1141 tcaagaacgg cacctctgat gtgttactgt gtgggaacag ctctgacgct gggacatgtc 1201 cggagggcta ccggtgccta aaggcaggcg agaaccccga ccacggctac accagcttcg 1261 attcctttgc ctgggccttt cttgcactct tccgcctgat gacgcaggac tgctgggagc 1321 gcctctatca gcagaccctc aggtccgcag ggaagatcta catgatcttc ttcatgcttg 1381 tcatcttcct ggggtccttc tacctggtga acctgatcct ggccgtggtc gcaatggcct 1441 atgaggagca aaaccaagcc accatcgctg agaccgagga gaaggaaaag cgcttccagg 1501 aggccatgga aatgctcaag aaagaacacg aggccctcac catcaggggt gtggataccg 1561 tgtcccgtag ctccttggag atgtcccctt tggccccagt aaacagccat gagagaagaa 1621 gcaagaggag aaaacggatg tcttcaggaa ctgaggagtg tggggaggac aggctcccca 1681 agtctgactc agaagatggt cccagagcaa tgaatcatct cagcctcacc cgtggcctca 1741 gcaggacttc tatgaagcca cgttccagcc gcgggagcat tttcaccttt cgcaggcgag 1801 acctgggttc tgaagcagat tttgcagatg atgaaaacag cacagcgggg gagagcgaga 1861 gccaccacac atcactgctg gtgccctggc ccctgcgccg gaccagtgcc cagggacagc 1921 ccagtcccgg aacctcggct cctggccacg ccctccatgg caaaaagaac agcactgtgg 1981 actgcaatgg ggtggtctca ttactggggg caggcgaccc agaggccaca tccccaggaa 2041 gccacctcct ccgccctgtg atgctagagc acccgccaga cacgaccacg ccatcggagg 2101 agccaggcgg gccccagatg ctgacctccc aggctccgtg tgtagatggc ttcgaggagc 2161 caggagcacg gcagcgggcc ctcagcgcag tcagcgtcct caccagcgca ctggaagagt 2221 tagaggagtc tcgccacaag tgtccaccat gctggaaccg tctcgcccag cgctacctga 2281 tctgggagtg ctgcccgctg tggatgtcca tcaagcaggg agtgaagttg gtggtcatgg 2341 acccgtttac tgacctcacc atcactatgt gcatcgtact caacacactc ttcatggcgc 2401 tggagcacta caacatgaca agtgaattcg aggagatgct gcaggtcgga aacctggtct 2461 tcacagggat tttcacagca gagatgacct tcaagatcat tgccctcgac ccctactact 2521 acttccaaca gggctggaac atcttcgaca gcatcatcgt catccttagc ctcatggagc 2581 tgggcctgtc ccgcatgagc aacttgtcgg tgctgcgctc cttccgcctg ctgcgggtct 2641 tcaagctggc caaatcatgg cccaccctga acacactcat caagatcatc gggaactcag 2701 tgggggcact ggggaacctg acactggtgc tagccatcat cgtgttcatc tttgctgtgg 2761 tgggcatgca gctctttggc aagaactact cggagctgag ggacagcgac tcaggcctgc 2821 tgcctcgctg gcacatgatg gacttctttc atgccttcct catcatcttc cgcatcctct 2881 gtggagagtg gatcgagacc atgtgggact gcatggaggt gtcggggcag tcattatgcc 2941 tgctggtctt cttgcttgtt atggtcattg gcaaccttgt ggtcctgaat ctcttcctgg 3001 ccttgctgct cagctccttc agtgcagaca acctcacagc ccctgatgag gacagagaga 3061 tgaacaacct ccagctggcc ctggcccgca tccagagggg cctgcgcttt gtcaagcgga 3121 ccacctggga tttctgctgt ggtctcctgc ggcagcggcc tcagaagccc gcagcccttg 3181 ccgcccaggg ccagctgccc agctgcattg ccacccccta ctccccgcca cccccagaga 3241 cggagaaggt gcctcccacc cgcaaggaaa cacggtttga ggaaggcgag caaccaggcc 3301 agggcacccc cggggatcca gagcccgtgt gtgtgcccat cgctgtggcc gagtcagaca 3361 cagatgacca agaagaagat gaggagaaca gcctgggcac ggaggaggag tccagcaagc 3421 agcaggaatc ccagcctgtg tccggtggcc cagaggcccc tccggattcc aggacctgga 3481 gccaggtgtc agcgactgcc tcctctgagg ccgaggccag tgcatctcag gccgactggc 3541 ggcagcagtg gaaagcggaa ccccaggccc cagggtgcgg tgagacccca gaggacagtt 3601 gctccgaggg cagcacagca gacatgacca acaccgctga gctcctggag cagatccctg 3661 acctcggcca ggatgtcaag gacccagagg actgcttcac tgaaggctgt gtccggcgct 3721 gtccctgctg tgcggtggac accacacagg ccccagggaa ggtctggtgg cggttgcgca 3781 agacctgcta ccacatcgtg gagcacagct ggttcgagac attcatcatc ttcatgatcc 3841 tactcagcag tggagcgctg gccttcgagg acatctacct agaggagcgg aagaccatca 3901 aggttctgct tgagtatgcc gacaagatgt tcacatatgt cttcgtgctg gagatgctgc 3961 tcaagtgggt ggcctacggc ttcaagaagt acttcaccaa tgcctggtgc tggctcgact 4021 tcctcatcgt agacgtctct ctggtcagcc tggtggccaa caccctgggc tttgccgaga 4081 tgggccccat caagtcactg cggacgctgc gtgcactccg tcctctgaga gctctgtcac 4141 gatttgaggg catgagggtg gtggtcaatg ccctggtggg cgccatcccg tccatcatga 4201 acgtcctcct cgtctgcctc atcttctggc tcatcttcag catcatgggc gtgaacctct 4261 ttgcggggaa gtttgggagg tgcatcaacc agacagaggg agacttgcct ttgaactaca
4321 ccatcgtgaa caacaagagc cagtgtgagt ccttgaactt gaccggagaa ttgtactgga
4381 ccaaggtgaa agtcaacttt gacaacgtgg gggccgggta cctggccctt ctgcaggtgg
4441 caacatttaa aggctggatg gacattatgt atgcagctgt ggactccagg gggtatgaag
4501 agcagcctca gtgggaatac aacctctaca tgtacatcta ttttgtcatt ttcatcatct
4561 ttgggtcttt cttcaccctg aacctcttta ttggtgtcat cattgacaac ttcaaccaac
4621 agaagaaaaa gttagggggc caggacatct tcatgacaga ggagcagaag aagtactaca
4681 atgccatgaa gaagctgggc tccaagaagc cccagaagcc catcccacgg cccctgaaca
4741 agtaccaggg cttcatattc gacattgtga ccaagcaggc ctttgacgtc accatcatgt
4801 ttctgatctg cttgaatatg gtgaccatga tggtggagac agatgaccaa agtcctgaga
4861 aaatcaacat cttggccaag atcaacctgc tctttgtggc catcttcaca ggcgagtgta
4921 ttgtcaagct ggctgccctg cgccactact acttcaccaa cagctggaat atcttcgact
4981 tcgtggttgt catcctctcc atcgtgggca ctgtgctctc ggacatcatc cagaagtact
5041 tcttctcccc gacgctcttc cgagtcatcc gcctggcccg aataggccgc atcctcagac
5101 tgatccgagg ggccaagggg
(SEQ ID NO: 10)
E28D SCN5A Splice Variant Complete Amino Acid Sequence (the portion of the sequence that is different from wild-type is bolded and underlined):
1 manfllprgt ssfrrftres laaiekrmae kqargsttlq esreglpeee aprpqldlqa
61 skklpdlygn ppqeligepl edldpfystq ktfivlnkgk tifrfsatna lyvlspfhpi
121 rraavkilvh slfnmlimct iltncvfmaq hdpppwtkyv eytftaiytf eslvkilarg
181 fclhaftflr dpwnwldfsv iimayttefv dlgnvsalrt frvlralkti svisglktiv
241 galiqsvkkl advmvltvfc lsvfaliglq lfmgnlrhkc vrnftalngt ngsveadglv
301 wesldlylsd penyllkngt sdvllcgnss dagtcpegyr clkagenpdh gytsfdsfaw
361 aflalfrlmt qdcwerlyqq tlrsagkiym iffmlviflg sfylvnlila vamayeeqn
421 qatiaeteek ekrfqeamem lkkehealti rgvdtvsrss lemsplapvn sherrskrrk
481 rmssgteecg edrlpksdse dgpramnhls ltrglsrtsm kprssrgsif tfrrrdlgse
541 adfaddenst ageseshhts llvpwplrrt saqgqpspgt sapghalhgk knstvdcngv
601 vsllgagdpe atspgshllr pvmlehppdt ttpseepggp qmltsqapcv dgfeepgarq
661 ralsavsvlt saleeleesr hkcppcwnrl aqryliwecc plwmsikqgv klvvmdpftd
721 ltitmcivln tlfmalehyn mtsefeemlq vgnlvftgif taemtfkiia ldpyyyfqqg
781 wnifdsiivi lslmelglsr msnlsvlrsf rllrvfklak swptlntlik iignsvgalg
841 nltlvlaiiv fifavvgmql fgknyselrd sdsgllprwh mmdffhafli ifrilcgewi
901 etmwdcmevs gqslcllvfl lvmvignlvv lnlflallls sfsadnltap dedremnnlq
961 lalariqrgl rfvkrttwdf ccgllrqrpq kpaalaaqgq lpsciatpys ppppetekvp
1021 ptrketrfee geqpgqgtpg dpepvcvpia vaesdtddqe edeenslgte eesskqqesq
1081 pvsggpeapp dsrtwsqvsa tasseaeasa sqadwrqqwk aepqapgcge tpedscsegs
1141 tadmtntael leqipdlgqd vkdpedcfte gcvrrcpcca vdttqapgkv wwrlrktcyh
1201 ivehswfetf iifmillssg alafediyle erktikvlle yadkmftyvf vlemllkwva
1261 ygfkkyftna wcwldflivd vslvslvant Igfaemgpik slrtlralrp Iralsrfegm
1321 rvvvnalvga ipsimnvllv clifwlifsi mgvnlfagkf grcinqtegd lplnytivnn
1381 ksqceslnlt gelywtkvkv nfdnvgagyl allqvatfkg wmdimyaavd srgyeeqpqw
1441 eynlymyiyf vifiifgsff tlnlfigvii dnfnqqkkkl ggqdifmtee qkkyynamkk
1501 lgskkpqkpi prplnkyqgf ifdivtkqaf dvtimflicl nmvtmmvetd dqspekinil
1561 akinllfvai ftgecivkla alrhyyftns wnifdfvvvi lsivgtylsd iiqkyffspt 1621 lfrvirlari grilrlirga kg
(SEQ ID NO: 11) A full-length SCN5A amino acid sequence is shown below.
1 manfllprgt ssfrrftres laaiekrmae kqargsttlq esreglpeee aprpqldlqa 61 skklpdlygn ppqeligepl edldpfystq ktfivlnkgk tifrfsatna lyvlspfhpi 121 rraavkilvh slfnmlimct iltncvfmaq hdpppwtkyv eytftaiytf eslvkilarg 181 fclhaftflr dpwnwldfsv iimayttefv dlgnvsalrt frvlralkti svisglktiv 241 galiqsvkkl advmvltvfc lsvfaliglq lfmgnlrhkc vrnftalngt ngsveadglv 301 wesldlylsd penyllkngt sdvllcgnss dagtcpegyr clkagenpdh gytsfdsfaw 361 aflalfrlmt qdcwerlyqq tlrsagkiym iffmlviflg sfylvnlila vamayeeqn 421 qatiaeteek ekrfqeamem lkkehealti rgvdtvsrss lemsplapvn sherrskrrk 481 rmssgteecg edrlpksdse dgpramnhls ltrglsrtsm kprssrgsif tfrrrdlgse 541 adfaddenst ageseshhts llvpwplrrt saqgqpspgt sapghalhgk knstvdcngv 601 vsllgagdpe atspgshllr pvmlehppdt ttpseepggp qmltsqapcv dgfeepgarq 661 ralsavsvlt saleeleesr hkcppcwnrl aqryliwecc plwmsikqgv kl vmdpftd 721 ltitmcivln tlfmalehyn mtsefeemlq vgnlvftgif taemtfkiia ldpyyyfqqg 781 wnifdsiivi lslmelglsr msnlsvlrsf rllrvfklak swptlntlik iignsvgalg 841 nltlvlaiiv fifa vgmql fgknyselrd sdsgllprwh mmdffhafli ifrilcgewi 901 etmwdcmevs gqslcllvfl lvmvignl v lnlflallls sfsadnltap dedremnnlq 961 lalariqrgl rfvkrttwdf ccgllrqrpq kpaalaaqgq lpsciatpys ppppetekvp 1021 ptrketrfee geqpgqgtpg dpepvcvpia vaesdtddqe edeenslgte eesskqqesq 1081 pvsggpeapp dsrtwsqvsa tasseaeasa sqadwrqqwk aepqapgcge tpedscsegs 1141 tadmtntael leqipdlgqd vkdpedcfte gcvrrcpcca vdttqapgkv wwrlrktcyh 1201 ivehswfetf iifmillssg alafediyle erktikvlle yadkmftyvf vlemllkwva 1261 ygfkkyftna wcwldflivd vslvslvant Igfaemgpik slrtlralrp Iralsrfegm 1321 rvvvnalvga ipsimnvllv clifwlifsi mgvnlfagkf grcinqtegd lplnytivnn 1381 ksqceslnlt gelywtkvkv nfdnvgagyl allqvatfkg wmdimyaavd srgyeeqpqw 1441 eynlymyiyf vifiifgsff tlnlfigvii dnfnqqkkkl ggqdifmtee qkkyynamkk 1501 lgskkpqkpi prplnkyqgf ifdivtkqaf dvtimflicl nmvtmmvetd dqspekinil 1561 akinllfvai ftgecivkla alrhyyftns wnifdfvvvi lsivgtvlsd iiqkyffspt 1621 lfrvirlari grilrlirga kgirtllfal mmslpalfni glllflvmfi ysifgmanfa 1681 yvkweagidd mfnfqtfans mlclfqitts agwdgllspi lntgppycdp tlpnsngsrg 1741 dcgspavgil ffttyiiisf livvnmyiai ilenfsvate esteplsedd fdmfyeiwek 1801 fdpeatqfie ysvlsdfada lseplriakp nqislinmdl pmvsgdrihc mdilfaftkr 1861 vlgesgemda lkiqmeekfm aanpskisye pitttlrrkh eevsamviqr afrrhllqrs 1921 lkhasflfrq qagsglseed aperegliay vmsenfsrpl gppssssiss tsfppsydsv 1981 tratsdnlqv rgsdyshsed ladfppspdr dresiv
(SEQ ID NO: 12) GenBank Accession NP_932173.1 (GI: 37622907), incorporated herein by reference.
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).
A full-length SCN5A nucleotide sequence is shown below. The start and stop codons of the coding sequence are bold and underlined. 1 agacggcggc ggcgcccgta ggatgcaggg atcgctcccc cggggccgct gagcctgcgc
61 ccagtgcccc gagccccgcg ccgagccgag tccgcgccaa gcagcagccg cccaccccgg 121 ggcccggccg ggggaccagc agcttcccca caggcaacgt gaggagagcc tgtgcccaga 181 agcaggatga gaagatggca aacttcctat tacctcgggg caccagcagc ttccgcaggt 241 tcacacggga gtccctggca gccatcgaga agcgcatggc agagaagcaa gcccgcggct 301 caaccacctt gcaggagagc cgagaggggc tgcccgagga ggaggctccc cggccccagc 361 tggacctgca ggcctccaaa aagctgccag atctctatgg caatccaccc caagagctca 421 tcggagagcc cctggaggac ctggacccct tctatagcac ccaaaagact ttcatcgtac 481 tgaataaagg caagaccatc ttccggttca gtgccaccaa cgccttgtat gtcctcagtc 541 ccttccaccc catccggaga gcggctgtga agattctggt tcactcgctc ttcaacatgc 601 tcatcatgtg caccatcctc accaactgcg tgttcatggc ccagcacgac cctccaccct 661 ggaccaagta tgtcgagtac accttcaccg ccatttacac ctttgagtct ctggtcaaga 721 ttctggctcg aggcttctgc ctgcacgcgt tcactttcct tcgggaccca tggaactggc 781 tggactttag tgtgattatc atggcataca caactgaatt tgtggacctg ggcaatgtct 841 cagccttacg caccttccga gtcctccggg ccctgaaaac tatatcagtc atttcagggc 901 tgaagaccat cgtgggggcc ctgatccagt ctgtgaagaa gctggctgat gtgatggtcc 961 tcacagtctt ctgcctcagc gtctttgccc tcatcggcct gcagctcttc atgggcaacc 1021 taaggcacaa gtgcgtgcgc aacttcacag cgctcaacgg caccaacggc tccgtggagg 1081 ccgacggctt ggtctgggaa tccctggacc tttacctcag tgatccagaa aattacctgc 1141 tcaagaacgg cacctctgat gtgttactgt gtgggaacag ctctgacgct gggacatgtc 1201 cggagggcta ccggtgccta aaggcaggcg agaaccccga ccacggctac accagcttcg 1261 attcctttgc ctgggccttt cttgcactct tccgcctgat gacgcaggac tgctgggagc 1321 gcctctatca gcagaccctc aggtccgcag ggaagatcta catgatcttc ttcatgcttg 1381 tcatcttcct ggggtccttc tacctggtga acctgatcct ggccgtggtc gcaatggcct 1441 atgaggagca aaaccaagcc accatcgctg agaccgagga gaaggaaaag cgcttccagg 1501 aggccatgga aatgctcaag aaagaacacg aggccctcac catcaggggt gtggataccg 1561 tgtcccgtag ctccttggag atgtcccctt tggccccagt aaacagccat gagagaagaa 1621 gcaagaggag aaaacggatg tcttcaggaa ctgaggagtg tggggaggac aggctcccca 1681 agtctgactc agaagatggt cccagagcaa tgaatcatct cagcctcacc cgtggcctca 1741 gcaggacttc tatgaagcca cgttccagcc gcgggagcat tttcaccttt cgcaggcgag 1801 acctgggttc tgaagcagat tttgcagatg atgaaaacag cacagcgggg gagagcgaga 1861 gccaccacac atcactgctg gtgccctggc ccctgcgccg gaccagtgcc cagggacagc 1921 ccagtcccgg aacctcggct cctggccacg ccctccatgg caaaaagaac agcactgtgg 1981 actgcaatgg ggtggtctca ttactggggg caggcgaccc agaggccaca tccccaggaa 2041 gccacctcct ccgccctgtg atgctagagc acccgccaga cacgaccacg ccatcggagg 2101 agccaggcgg gccccagatg ctgacctccc aggctccgtg tgtagatggc ttcgaggagc 2161 caggagcacg gcagcgggcc ctcagcgcag tcagcgtcct caccagcgca ctggaagagt 2221 tagaggagtc tcgccacaag tgtccaccat gctggaaccg tctcgcccag cgctacctga 2281 tctgggagtg ctgcccgctg tggatgtcca tcaagcaggg agtgaagttg gtggtcatgg 2341 acccgtttac tgacctcacc atcactatgt gcatcgtact caacacactc ttcatggcgc 2401 tggagcacta caacatgaca agtgaattcg aggagatgct gcaggtcgga aacctggtct 2461 tcacagggat tttcacagca gagatgacct tcaagatcat tgccctcgac ccctactact 2521 acttccaaca gggctggaac atcttcgaca gcatcatcgt catccttagc ctcatggagc 2581 tgggcctgtc ccgcatgagc aacttgtcgg tgctgcgctc cttccgcctg ctgcgggtct 2641 tcaagctggc caaatcatgg cccaccctga acacactcat caagatcatc gggaactcag 2701 tgggggcact ggggaacctg acactggtgc tagccatcat cgtgttcatc tttgctgtgg 2761 tgggcatgca gctctttggc aagaactact cggagctgag ggacagcgac tcaggcctgc 2821 tgcctcgctg gcacatgatg gacttctttc atgccttcct catcatcttc cgcatcctct 2881 gtggagagtg gatcgagacc atgtgggact gcatggaggt gtcggggcag tcattatgcc 2941 tgctggtctt cttgcttgtt atggtcattg gcaaccttgt ggtcctgaat ctcttcctgg 3001 ccttgctgct cagctccttc agtgcagaca acctcacagc ccctgatgag gacagagaga 3061 tgaacaacct ccagctggcc ctggcccgca tccagagggg cctgcgcttt gtcaagcgga 3121 ccacctggga tttctgctgt ggtctcctgc ggcagcggcc tcagaagccc gcagcccttg 3181 ccgcccaggg ccagctgccc agctgcattg ccacccccta ctccccgcca cccccagaga 3241 cggagaaggt gcctcccacc cgcaaggaaa cacggtttga ggaaggcgag caaccaggcc 3301 agggcacccc cggggatcca gagcccgtgt gtgtgcccat cgctgtggcc gagtcagaca 3361 cagatgacca agaagaagat gaggagaaca gcctgggcac ggaggaggag tccagcaagc 3421 agcaggaatc ccagcctgtg tccggtggcc cagaggcccc tccggattcc aggacctgga 3481 gccaggtgtc agcgactgcc tcctctgagg ccgaggccag tgcatctcag gccgactggc 3541 ggcagcagtg gaaagcggaa ccccaggccc cagggtgcgg tgagacccca gaggacagtt 3601 gctccgaggg cagcacagca gacatgacca acaccgctga gctcctggag cagatccctg 3661 acctcggcca ggatgtcaag gacccagagg actgcttcac tgaaggctgt gtccggcgct 3721 gtccctgctg tgcggtggac accacacagg ccccagggaa ggtctggtgg cggttgcgca 3781 agacctgcta ccacatcgtg gagcacagct ggttcgagac attcatcatc ttcatgatcc 3841 tactcagcag tggagcgctg gccttcgagg acatctacct agaggagcgg aagaccatca 3901 aggttctgct tgagtatgcc gacaagatgt tcacatatgt cttcgtgctg gagatgctgc 3961 tcaagtgggt ggcctacggc ttcaagaagt acttcaccaa tgcctggtgc tggctcgact 4021 tcctcatcgt agacgtctct ctggtcagcc tggtggccaa caccctgggc tttgccgaga 4081 tgggccccat caagtcactg cggacgctgc gtgcactccg tcctctgaga gctctgtcac 4141 gatttgaggg catgagggtg gtggtcaatg ccctggtggg cgccatcccg tccatcatga 4201 acgtcctcct cgtctgcctc atcttctggc tcatcttcag catcatgggc gtgaacctct 4261 ttgcggggaa gtttgggagg tgcatcaacc agacagaggg agacttgcct ttgaactaca 4321 ccatcgtgaa caacaagagc cagtgtgagt ccttgaactt gaccggagaa ttgtactgga 4381 ccaaggtgaa agtcaacttt gacaacgtgg gggccgggta cctggccctt ctgcaggtgg 4441 caacatttaa aggctggatg gacattatgt atgcagctgt ggactccagg gggtatgaag 4501 agcagcctca gtgggaatac aacctctaca tgtacatcta ttttgtcatt ttcatcatct 4561 ttgggtcttt cttcaccctg aacctcttta ttggtgtcat cattgacaac ttcaaccaac 4621 agaagaaaaa gttagggggc caggacatct tcatgacaga ggagcagaag aagtactaca 4681 atgccatgaa gaagctgggc tccaagaagc cccagaagcc catcccacgg cccctgaaca 4741 agtaccaggg cttcatattc gacattgtga ccaagcaggc ctttgacgtc accatcatgt 4801 ttctgatctg cttgaatatg gtgaccatga tggtggagac agatgaccaa agtcctgaga 4861 aaatcaacat cttggccaag atcaacctgc tctttgtggc catcttcaca ggcgagtgta 4921 ttgtcaagct ggctgccctg cgccactact acttcaccaa cagctggaat atcttcgact 4981 tcgtggttgt catcctctcc atcgtgggca ctgtgctctc ggacatcatc cagaagtact 5041 tcttctcccc gacgctcttc cgagtcatcc gcctggcccg aataggccgc atcctcagac 5101 tgatccgagg ggccaagggg atccgcacgc tgctctttgc cctcatgatg tccctgcctg 5161 ccctcttcaa catcgggctg ctgctcttcc tcgtcatgtt catctactcc atctttggca 5221 tggccaactt cgcttatgtc aagtgggagg ctggcatcga cgacatgttc aacttccaga 5281 ccttcgccaa cagcatgctg tgcctcttcc agatcaccac gtcggccggc tgggatggcc 5341 tcctcagccc catcctcaac actgggccgc cctactgcga ccccactctg cccaacagca 5401 atggctctcg gggggactgc gggagcccag ccgtgggcat cctcttcttc accacctaca 5461 tcatcatctc cttcctcatc gtggtcaaca tgtacattgc catcatcctg gagaacttca 5521 gcgtggccac ggaggagagc accgagcccc tgagtgagga cgacttcgat atgttctatg 5581 agatctggga gaaatttgac ccagaggcca ctcagtttat tgagtattcg gtcctgtctg 5641 actttgccga tgccctgtct gagccactcc gtatcgccaa gcccaaccag ataagcctca 5701 tcaacatgga cctgcccatg gtgagtgggg accgcatcca ttgcatggac attctctttg 5761 ccttcaccaa aagggtcctg ggggagtctg gggagatgga cgccctgaag atccagatgg 5821 aggagaagtt catggcagcc aacccatcca agatctccta cgagcccatc accaccacac 5881 tccggcgcaa gcacgaagag gtgtcggcca tggttatcca gagagccttc cgcaggcacc 5941 tgctgcaacg ctctttgaag catgcctcct tcctcttccg tcagcaggcg ggcagcggcc 6001 tctccgaaga ggatgcccct gagcgagagg gcctcatcgc ctacgtgatg agtgagaact 6061 tctcccgacc ccttggccca ccctccagct cctccatctc ctccacttcc ttcccaccct 6121 cctatgacag tgtcactaga gccaccagcg ataacctcca ggtgcggggg tctgactaca 6181 gccacagtga agatctcgcc gacttccccc cttctccgga cagggaccgt gagtccatcg 6241 tgtgagcctc ggcctggctg gccaggacac actgaaaagc agcctttttc accatggcaa 6301 acctaaatgc agtcagtcac aaaccagcct ggggccttcc tggctttggg agtaagaaat 6361 gggcctcagc cccgcggatc aaccaggcag agttctgtgg cgccgcgtgg acagccggag 6421 cagttggcct gtgcttggag gcctcagata gacctgtgac ctggtctggt caggcaatgc 6481 cctgcggctc tggaaagcaa cttcatccca gctgctgagg cgaaatataa aactgagact 6541 gtatatgttg tgaatgggct ttcataaatt tattatattt gatatttttt tacttgagca 6601 aagaactaag gatttttcca tggacatggg cagcaattca cgctgtctct tcttaaccct 6661 gaacaagagt gtctatggag cagccggaag tctgttctca aagcagaagt ggaatccagt 6721 gtggctccca caggtcttca ctgcccaggg gtcgaatggg gtccccctcc cacttgacct 6781 gagatgctgg gagggctgaa cccccactca cacaagcaca cacacacagt cctcacacac 6841 ggaggccaga cacaggccgt gggacccagg ctcccagcct aagggagaca ggcctttccc 6901 tgccggcccc ccaaggatgg ggttcttgtc cacggggctc actctggccc cctattgtct 6961 ccaaggtccc attttccccc tgtgttttca cgcaggtcat attgtcagtc ctacaaaaat 7021 aaaaggcttc cagaggagag tggcctgggt cccagggctg gccctaggca ctgatagttg 7081 ccttttcttc ccctcctgta agagtattaa caaaaccaaa ggacacaagg gtgcaagccc 7141 cattcacggc ctggcatgca gcttgtcctt gctcctggaa cctggcaggc cctgcccagc 7201 cagccatcgg aagagagggc tgagccatgg gggtttgggg ctaagaagtt caccagccct 7261 gagccatggc ggcccctcag cctgcctgaa gagaggaaac tggcgatctc ccagggctct 7321 ctggaccata cgcggaggag ttttctgtgt ggtctccagc tcctctccag acacagagac 7381 atgggagtgg ggagcggagc ttggccctgc gccctgtgca gggaaaggga tggtcaggcc 7441 cagttctcgt gcccttagag gggaatgaac catggcacct ttgagagagg gggcactgtg 7501 gtcaggccca gcctctctgg ctcagcccgg gatcctgatg gcacccacac agaggacctc 7561 tttggggcaa gatccaggtg gtcccatagg tcttgtgaaa aggctttttc agggaaaaat 7621 attttactag tccaatcacc cccaggacct cttcagctgc tgacaatcct atttagcata 7681 tgcaaatctt ttaacataga gaactgtcac cctgaggtaa cagggtcaac tggcgaagcc 7741 tgagcaggca ggggcttggc tgccccattc cagctctccc atggagcccc tccaccgggc 7801 gcatgcctcc caggccacct cagtctcacc tgccggctct gggctggctg ctcctaacct 7861 acctcgccga gctgtcggag ggctggacat ttgtggcagt gctgaagggg gcattgccgg 7921 cgagtaaagt attatgtttc ttcttgtcac cccagttccc ttggtggcaa ccccagaccc 7981 aacccatgcc cctgacagat ctagttctct tctcctgtgt tccctttgag tccagtgtgg 8041 gacacggttt aactgtccca gcgacatttc tccaagtgga aatcctattt ttgtagatct 8101 ccatgctttg ctctcaaggc ttggagaggt atgtgcccct cctgggtgct caccgcctgc 8161 tacacaggca ggaatgcggt tgggaggcag gtcgggctgc cagcccagct ggccggaagg 8221 agactgtggt ttttgtgtgt gtggacagcc cgggagcttt gagacaggtg cctggggctg 8281 gctgcagacg gtgtggttgg gggtgggagg tgagctagac ccaaccctta gcttttagcc 8341 tggctgtcac ctttttaatt tccagaactg cacaatgacc agcaggaggg aaggacagac 8401 atcaagtgcc agatgttgtc tgaactaatc gagcacttct caccaaactt catgtataaa 8461 taaaatacat atttttaaaa caaaccaata aatggcttac atga
(SEQ ID NO: 13) GenBank Accession NM_198056.2 (GI: 124518659), incorporated herein by reference.
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
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. Each HU protein has three RNA recognition motifs (RRMs 1-3), which share more than 90% amino acid sequence identity among family members. 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.
Additionally, 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." Thus, as RBPs, 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.
Although 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.
The HU proteins: HuA (HuR in Human), HuB (HelNl in Human), HuC, and HuD are a family of mammalian RNA-binding proteins (Zhu et al., 2006, Mol. Biol. Cell. 17: 5105-5114). Of the four Hu family members, HuA/R is widely expressed in many cell types, whereas HuB, HuC, and HuD, are expressed specifically in neurons. In addition to HuR (ELAVL1, GenBank accession number: NM_001419.2), the ELAV/HU family members HuB (ELAVL2, GenBank accession number: NM_004432.3), HuC (ELAVL3, GenBank accession number: NM_001420.3) and HuD (ELAVL4, GenBank accession number: NM_001144774.1) are also useful diagnostic or prognostic markers.
HuR and HuB protein
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.
The initiator methionine of HuR is removed in the mature processed form yielding a mature protein of 325 amino acids. 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); and the RRM3 domain comprises residues 244-322 (79 amino acids in length). Fragments of HuR, such as fragments
corresponding to the domains described above, are useful in the compositions and methods described herein. The protein is characterized by the following amino acid modifications:
Modified residue 2-N-acetylserine; modified residue 2-Phospho serine; modified residue 202- Phosphoserine, and/or modified residue 217-Omega-N-methylated arginine. This encoded protein contains 3 RNA-binding domains and binds cis-acting AU-rich elements. It stabilizes mRNAs and thereby regulates gene expression.
Human HuR amino acid sequence
1 msngyedhma edcrgdigrt nlivnylpqn mtqdelrslf ssigevesak lirdkvaghs 61 lgygfvnyvt akdaeraint lnglrlqskt ikvsyarpss evikdanlyi sglprtmtqk 121 dvedmfsrfg riinsrvlvd qttglsrgva firfdkrsea eeaitsfngh kppgssepit 181 vkfaanpnqn knvallsqly hsparrfggp vhhqaqrfrf spmgvdhmsg lsgvnvpgna 241 ssgwcifiyn lgqdadegil wqmfgpfgav tnvkvirdfn tnkckgfgfv tmtnyeeaam 301 aiaslngyrl gdkilqvsfk tnkshk
(SEQ ID NO: 14) 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 (β-strand region), residues 157-167 (helical region), resiudes 146-156 (β-strand region), residues 141-143
(hydrogen bonded turn), residues 107-185 (RNA binding site), residues 106-189 (RNA recognition motif), residues 19-96 (RNA recognition motif 1), residues 19-326 (ELAV/HuD family splicing factor).
Human HuR nucleotide sequence. The start and stop codons of the coding sequence are bold and underlined.
1 ggtcgtgcgc gctgaggagg agccgctgcc gccgtcgccg tcgccgccac cgccgccacc 61 gctaccgagg ccgagcggag ccgttagcgc cgcgccgccg ccgcctcccg cccgccccgg 121 agcagccccg ggcccgcccg cccgcatcca gatttttgaa aaatacaatg tctaatggtt 181 atgaagacca catggccgaa gactgcaggg gtgacatcgg gagaacgaat ttgatcgtca 241 actacctccc tcagaacatg acccaggatg agttacgaag cctgttcagc agcattggtg 301 aagttgaatc tgcaaaactt attcgggata aagtagcagg acacagcttg ggctatggct 361 ttgtgaacta cgtgaccgcg aaggatgcag agagagcgat caacacgctg aacggcttga 421 ggctccagtc aaaaaccatt aaggtgtcgt atgctcgccc gagctcagag gtgatcaaag 481 acgccaactt gtacatcagc gggctcccgc ggaccatgac ccagaaggac gtagaagaca 541 tgttctctcg gtttgggcgg atcatcaact cgcgggtcct cgtggatcag actacaggtt 601 tgtccagagg ggttgcgttt atccggtttg acaaacggtc ggaggcagaa gaggcaatta 661 ccagtttcaa tggtcataaa cccccaggtt cctctgagcc catcacagtg aagtttgcag 721 ccaaccccaa ccagaacaaa aacgtggcac tcctctcgca gctgtaccac tcgccagcgc 781 gacggttcgg aggccccgtt caccaccagg cgcagagatt caggttctcc cccatgggcg 841 tcgatcacat gagcgggctc tctggcgtca acgtgccagg aaacgcctcc tccggctggt 901 gcattttcat ctacaacctg gggcaggatg ccgacgaggg gatcctctgg cagatgtttg 961 ggccgtttgg tgccgtcacc aatgtgaaag tgatccgcga cttcaacacc aacaagtgca 1021 aagggtttgg ctttgtgacc atgacaaact atgaagaagc cgcgatggcc atagccagcc 1081 tgaacggcta ccgcctgggg gacaaaatct tacaggtttc cttcaaaacc aacaagtccc 1141 acaaataact cgctcatgct tttttttgta cggaatagat aattaagagt gaaggagttg 1201 aaacttttct tgttagtgta caactcattt tgcgccaatt ttcacaagtg tttgtctttg 1261 tctgaatgag aagtgagaag gtttttatac tctgggatgc aaccgacatg ttcaaatgtt 1321 tgaaatccca caatgttaga ccaatcttaa gtttcgtaag ttatttcctt taagatatat 1381 attaaacaga aatctaagta gaactgcatt gactaaccag tccctctgga tggtggtgaa 1441 cctgaagcat gctttaacct ctaagactgt ctaacacgcg tttcattcaa tgtctccaca 1501 gactgggtag caaaaaaatc accttttagt tttagttttt aatctaaaga tgttagacag 1561 atgctgagtg tgcgttttct caaccgcttc aacattgtaa gcgatgtatg ctttggttga 1621 caggaagttc cttttccagg caggtcccgt tgccacctcc tgctcactca gtcccgggct 1681 ctgccgagtg gtcctgggaa tggcggcggg cccgtccagc gtgggccacc actggggccg 1741 ggggccacgg gctgcatgct gggcgggccc tccagagaag gacacaaacg tgtttcgtaa 1801 gcccaggcac caatgggaat ggaccaaaga gtttcaggga aactccagta tattccagag 1861 tcagatctaa gctccaggca cgcctgaaga tgtgttgcta ctctgacatc ccgagtttct 1921 gtccacacat tgcatgcaca gcgccccaca cattggatac tgttgttcac gataatttct 1981 cccgttttcc agagcattta acatagcttg gaggcgtaaa atggctctgt attttaataa 2041 cacagaaaca tttgagcatt gtatttctcg catcccttct cgtgagcgct tagacctttt 2101 tctattttag tcggattttg ttttggaatt ttgcttttgt atgaacactc agcagaaaag 2161 tacttacttc ttgccagtta tctattaacc aaaacctttg atttgtagtt ttaaagatta 2221 accctcaaag ttctcttcat aactgccttg acattttggg ttgttctgtt ctgttaattt 2281 tcttttgctt ttttgtgttt tttgtttgtt tttacttttg catttaagac cattaaattt 2341 gattttgttt tgctcgaatt ttgttttgtt ttttatttta ccttttcttt ctttttggct 2401 agggaaggtg caggtggccc agcattcagg gaggagtcgt aagatcttaa gaaaccaatc 2461 cttgcctcaa gcaaaagcat ttctgaatct gtgacgcaag aatgtgcagt tacaggctgg 2521 tggcttttaa accaggagcc cggaggaagg gtgaaagaga aagcctgtga aataggcagg 2581 gccagatcac ccaaaactcc tcaggactgg gatctggcgt ttataaataa ctagtttaca 2641 gagagaatca caaacaggat aacttagtac cagcagtttt taaccttgac gtgagactaa 2701 aacgtgaccg taggctgttt tttagttatt gctctcatga gatgatggct gtatttatct 2761 gtttatttat acctatttat gtatttattt attgaagtgt gaaattgagc aataggcagg 2821 caccaccgtc cccagagcag gtcagcgtct cgagaggccc ctggacaatt gaggatgccc 2881 atcccctccc ctttccctga tcttttactg aggggctgtg tgcgcgatcc ttgcaaattg 2941 atgatgttgc catccgtacc caggctgtgt ctcataaaag tcggcctggt gccagagagg 3001 acctcctttc tcccacagaa tcccagatcc tcaggaaaag ccaaaaccga ggcccattgc 3061 ccggattcga cacaaaagag ggtccctgct ctgttgcccg agagcagtct gcatcctggg 3121 accagaatgc tttcctggaa aaagaagcct ttcaggtttc cctgggccag catcttctga 3181 tggaaggtgg gagccaacac ccttctgatg gaaggtggga gccaacaccc ttctgatgga 3241 aggtgggagc caacaccctt ctgatggaag gtgggagcca acacccttct gatggaaggt 3301 gggagccaac acccttctga tggaaggcgg gattcccgct tctgaaactc cccctggagt 3361 ctcactccca cacatgccca tagctagcat tcaacagaga actctgtctt aagcttcaac 3421 tgtgaaaatg atgacgggct tgtagcacct cagcttcttt cctcgccccc ttttatctga 3481 atcctatcaa ttattctgat gctgggacag gtgagaagaa actgtgaagt atatgagcct 3541 ttgaaagttc cctgaagttt ctcagttcag gaacattctc attgtatgtg gtctccgctg 3601 tttgaacagc cttctagcta aaaaattcca aagcctttat ttgggagtct tagcttgcaa 3661 gcttgtggaa ggatttagct taacaactgt cactcctgaa aagcaatctc tgttccatca 3721 aggttctagt tgctggccct gtgtcctcaa agttcattac atcttatcaa ggcctgtttg 3781 caaaggggag atcccttttc ttaaaaaagg ctcaacccaa aagaaaccat ttcttaaaaa 3841 attttacata gatcagttgt atttctattt agcaaaaatg agtgctctgc ttttatttgg 3901 gaatttcgat gaaaaagcgt tcagagtaga taatgttcat ttatcaaaaa tctggtttgg 3961 gaaataccaa agaggctttg attgaattcc cttttgaccc gtgtgtaact tcctctggta 4021 gttagacccc aggcagctcc gaatttgtga acctgcttcc tgatgaattc tcccttgttc 4081 ccccttggct ctgccattat ttcgttttca gtgtaatttg ccaagccgca gttttctgtg 4141 ctggctgtgc ctctagtcgc agctctgtga ctgattccct cccgggtgct gagtcccctc 4201 cccggccacc atcctgcgtg aatatcctga aattcatggg cttcctcggg ggcccgcccg 4261 caggtggtgc tgggtgggtt ccgccacctt ctcctggaag gtgagccttt tcctggccaa 4321 gggcagctgc cttaacctct gagagtctgc gcttggcctt agtcctggag acccagcctc 4381 cagggactga accgtgctgc tgttgggagc caagaccggc cctttggagc cggcagccca 4441 ggggtccctg ctggatcaga gaaatagaag cacccgaaga cggttagtgg caattccttg 4501 acccggtttg cttccaaatg aaggccattt gtccaccagg cattgaaaag acatgactta 4561 cccagtccgg catcggactt gaaaaatcga aattgacatc actcagctgt tacatttcac 4621 atccgattca gccccctttt atttccatgt gcttttcgca gccttcctgt gttggatgaa 4681 agagaataag aattcagctg acaggaggcc tctatcctgt ccctccaccc caccctccac 4741 ctcaatcccc tcccatcttc cccagaccta cctcacctac taggacctga ggcagctcct 4801 tagcagagac ccctggggtt tagctgactc tgggggtcag gggttcctgt ccccaaactt 4861 cgcaagacag ccctgaagtc acaagtgctt tcttttaagt ggcattggca attggcgtgt 4921 aatgatggca gtagaatctg aatctcggat cccaggcagg gttcacattt ccaaaccttt 4981 ttgatttccc ctgacctcta atggctggat cctatttttc tacaaccttt cagtgacatc 5041 gttcaggttt ctttcttggc catttaaaaa aacaaatttt ttttttctca cttgtaagtc 5101 accgccagta cctaagttag gctaacggag actttgacag gactggattt ttcttccacc 5161 agaagagaag ccttttccgt tggtttgggg ccacctcttt gaccatgacc atgtgatgtt 5221 ccgtttacag tgacttgctt tgggggaggg gaggctctct taaccgattc ccatgttgta 5281 cagtagatgg ttagaccttt tgtatattag tgtgttttaa gttattgatt tgttttatat 5341 aaaataattt atttttcagg tgccattttt cattttaact ttgtttttac atgggtttgt 5401 tttcaataaa gtctgacact ggtgtccaaa agtcaacaat aaaatgaatc ccattgtgtt 5461 cttttgaaga tgcctatgta acttttaagc tttttaaatt attttcagaa aaaaaaaaag 5521 aaaagccctt atcagttttc catcagccca ttgccttttt attttttttt tttaatcctt 5581 gtgaataaat gttctttagt gttttaggag gaaaaagcaa acctagattt tgataaccca 5641 gaagacttca gattaataaa gaagctttga aagaagacca tttttcaaaa ttttagtgaa 5701 gtgtgaatat tttttgtcaa tggctttctc aaagagaatg aaacttttgc accattttca 5761 gagtttttat agagatgcca aattgatata tttacatgta atggaaacat gaaaaagttt 5821 tattaaacaa ttgttcatag ctgtgtagac attttaattc agtttccaaa gctctcaaaa
5881 aatcgtattt ttgaagtacg gagtgatgcg gtttggggcg tggcttacag ttccaacgac
5941 tcaattgtcc cgatactcag ttctttctac aggtatcagg ttcgtgttaa acgctgtatg
6001 ttaactatga ctggaattct gtgatatttt ggtaataaat gaagtgggat cattgcgaaa
6061 aaaaaaaaaa aaaaa
(SEQ ID NO: 15) GenBank accession number: NM_001419.2 (GI: 38201713), incorporated herein by reference.
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).
Human HuB amino acid sequence
1 metqlsngpt cnntangptt innncsspvd sgntedsktn livnylpqnm tqeelkslfg
61 sigeiesckl vrdkitgqsl gygfvnyidp kdaekaintl nglrlqtkti kvsyarpssa
121 sirdanlyvs glpktmtqke leqlfsqygr iitsrilvdq vtgisrgvgf irfdkrieae
181 eaikglngqk ppgatepitv kfannpsqkt nqailsqlyq spnrrypgpl aqqaqrfrld
241 nllnmaygvk rfspmtidgm tslaginipg hpgtgwcifv ynlapdades ilwqmfgpfg
301 avtnvkvird fntnkckgfg fvtmtnydea amaiaslngy rlgdrvlqvs fktnkthka
(SEQ ID NO: 16) GenBank Accession NP_004423.2 (GI: 115511032), incorporated herein by reference.
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.
Human HuB nucleotide sequence. The start and stop codon of the coding sequence are bold and underlined.
1 caataggagg gtagtctctc cgtcttttta aactcttttt taagtttccc ctcccctttc
61 atattttttt tcgccatttc ttttagcatt ggactttggg gtcgaaagcg tttcttttta
121 tttgcttctt ttaagccgag cacagtttag gtttcgtgct gtcttaagag aactatccag
181 cagcttcttg ctcatcctta ttgggagaac tgcaccgtta ctttaaaaac acacatacac
241 aaaaacctta agggagaaag caggtaattg ctgccatgga aacacaactg tctaatgggc
301 caacttgcaa taacacagcc aatggtccaa ccaccataaa caacaactgt tcgtcaccag
361 ttgactctgg gaacacagaa gacagcaaga ccaacttaat agtcaactac cttcctcaga
421 acatgacaca ggaggaacta aagagtctct ttgggagcat tggtgaaata gagtcctgta
481 agcttgtaag agacaaaata acagggcaga gcttgggata tggctttgtg aactacattg
541 accccaagga tgcagagaaa gctatcaaca ccctgaatgg attgagactt caaaccaaaa
601 caataaaagt ttcctatgct cgcccaagtt cagcttctat cagagatgca aatttatatg 661 tcagcggact tccaaaaaca atgacccaga aggagttgga acagcttttt tcacaatatg 721 gacgcattat tacttctcgt attcttgtcg accaggtcac tggcatatca aggggtgtag 781 ggtttattcg atttgacaag cgaattgagg cagaagaagc tatcaaaggc ctaaatggcc 841 agaaacctcc cggtgccacg gagccaatca ctgtaaagtt tgctaataac ccaagccaaa 901 aaaccaatca ggccatcctt tcccagctgt accagtctcc aaacagaagg tatccaggac 961 cgctagctca gcaggcacag cgttttaggt tggacaatct gctcaatatg gcttatggag 1021 taaagaggtt ttctccaatg accattgacg gaatgaccag tttggctgga attaatatcc 1081 ctgggcaccc tggaacaggg tggtgtatat ttgtgtacaa cctggctcct gacgcagatg 1141 agagtatcct gtggcaaatg tttgggcctt ttggagctgt caccaatgtg aaggtcatcc 1201 gtgactttaa caccaataaa tgcaaaggtt ttggatttgt gactatgaca aactatgatg 1261 aggctgccat ggcgatagct agcctcaatg gataccgtct gggagacaga gtactgcagg 1321 tctcctttaa gacaaacaaa acgcacaaag cctaatgagc tcttgtcctc agtccattta 1381 tatatgaaaa ctatacaaca aaggcaagtt aagagaaact ttatacatta gtaaatgtct 1441 ttgtaagtca gtgttgagat ggggataaaa tgactactta gcatcctaag aaatatgtga 1501 gattttttat tgctagtatt tgaattaaaa cttcttaaat atcttttatg tttgaatatg 1561 gacaagaggt acagggtttt tacctgtcac attgcattct attgccttct ttgaagaagg 1621 tggacctttt aaagtgtttc agctaaggga agacatttct tttcttttta cataactgcc 1681 ttgaacctgt gagtaaatat tgaggctttg tgttgtaatt cttcagttgg ttgtgtcttt 1741 tttttccccc ctttttttcc tttttctgat tagctttgtg tttggtttac atttaaagca 1801 ttgctgttat gtctgtttaa gaaaagtatt ttgaagttta catttttatt tatgaagttt 1861 aaaacagtat ttattttgta attatgattt gggttgggga agggggggct acattataaa 1921 cgcttattgt aagaatactg gagaactttt cgtaaagcag taccttgcca aagagataag 1981 agcctctttg atgtgggttt aaaaaaagca tctattttta taaaaaagaa aatttggaga 2041 aactttttac tggtcctgga acaaatattt tgacttgaat actttgagaa atctcttcat 2101 atgacaccta gtgagctttt aaaatttacc aggaaatttg cagcggttgg aaaatttaga 2161 aagatttatg gtgtagaaaa tacttttgag atctttgtat gaaaggagta gaatcaatgg 2221 ggggaaacac tgctggtttc atttttgtaa tcaccagtgg agcgtctgat catcctggtt 2281 attatgtgat aggtggctca cattgatttg tgattttgaa acaaataaaa aaaatttaca 2341 aaagaatata taagagcagg caagaaattt aaattaccga gagatggggg aaaaaatctg 2401 ttcttcctaa agaaatccct tcagatagag ctcatggtgt ttagtgatgt acttgcagta 2461 ttgtttgaag aattgttttg tcttaaggaa aaaagacgtt gcacatgatt tgtactgcag 2521 caaatcagca aaagtgatct gagttggata tatttgaagg tattttgaaa gttacgttca 2581 aggctaacac ctgagctttg tgtaatgtaa ataagacctt gtgtttatga acctttcagc 2641 taatttaatt ttttttccct tacatgccaa gtgatgttca ggttttgaat gtttttgtat 2701 cagttttttc ctttgtaaat ggcattaaca ttgttacttg aggtcttgct taatcacttt 2761 tgttgtcctg aggacttgaa tttacagtgc atcagatttg ttgcaaattt tgtctgtaga 2821 tagtctagct tcagctgttt atggtgatgc tacattttcg tttataaata tgtttgtggt 2881 ataaaaaaat gagtataacc ataggttttg aacaaatttc cttacatttt tcatacaaaa 2941 atcataaata tctgtatgct attgaaattt aactttgtat gatgcttaaa aaccactatt 3001 tggggaaata ataaaataag tctttaccat gtatgaaaga aattttaaaa aatacaaaat 3061 attttctgat tagcatctag cttataataa attttcaaaa aagctgaagg caaaaatgcc 3121 ttcatcagga tgcactgaga actatatagt tacgtcctgc tttttgtata aactgagatg 3181 ctcacatgct tccccttaga acaggcaatg tgctatgcat aacatagttg tacattatct 3241 ttgcggttgc tttgagtttt attttttatt atttaaaatt gtagttataa aatttttcag 3301 tatagtacag tacatatact gtgaggcgcg tgctaaagtg aataagcgag ttttcatgct 3361 gacccactca atgctattca gaaatcaatt ggcttagcac tttctcatat ccttaggtgc 3421 atttagattg ccagagttaa ccttctgcgt ttaaaaaaag aaaaacacta aaaaataaaa 3481 tacatgtata tacttaaaaa aaaataataa ggtttccctc aagggaaaac agcagctaca 3541 tgcttctttc ctatactact gtagcaaacc aaggcattga tgagagggca tgcaaattgt 3601 gcttcacttt acagtgtttt atcagagcac ttaataaaat gtaaggctgg tatttatttg 3661 aagttgtaca gtatgactta attcacatct gttggaatag aaaatatatt ctgttgagta 3721 tttaagaggc tgtacatgtt ttcttttgtg tttggattct ttgtactttt tcatgttcag 3781 tacatcaata aacaaagttg aagggaaaaa aaaa
(SEQ ID NO: 17) GenBank accession number: NM_004432.3 (GI: 283945527), incorporated herein by reference. Exemplary regions or fragments of HuB nucleic acid sequences include bases 936-938 (phosphorylation site), and 3787-3792 (regulator sequence, polyA signal sequence).
MEF2C (Myocyte- specific enhancer factor 2C)
MEF2C, also known as MADS box transcription enhancer factor 2, polypeptide c is involved in cardiac morphogenesis, myogenesis and vascular development. The myocyte enhancer factor-2 (MEF-2) family of transcription factors associate with co-repressors or co- activators to regulate development and function of T cells, neuronal cells, and muscle cells. Four family members arise from alternatively spliced transcripts, termed MEF2A, -2B, -2C, and -2D. These members bind as homo- and helerodimers to the MEF2 site in the promoter region of affected genes. Differential regulation in the expression of the four transcripts implies functional distinction for each during embryogenesis and development. 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. In adult tissues, Mef2 proteins regulate the sters-response during cardiac hypertrophy and tissue remodeling in cardiac and skeletal muscle.
Human MEF2C amino acid sequence
1 mgrkkiqitr imdernrqvt ftkrkfglmk kayelsvlcd ceialiifns tnklfqyast 61 dmdkvllkyt eynephesrt nsdivetlrk kglngcdspd pdaddsvghs pesedkyrki 121 nedidlmisr qrlcavpppn fempvsipvs shnslvysnp vsslgnpnll plahpslqrn 181 smspgvthrp psagntgglm ggdltsgagt sagngygnpr nspgllvspg nlnknmqaks 241 pppmnlgmnn rkpdlrvlip pgskntmpsv sedvdlllnq rinnsqsaqs latp vsvat 301 ptlpgqgmgg ypsaisttyg teyslssadl sslsgfntas alhlgsvtgw qqqhlhnmpp 361 salsqlgact sthlsqssnl slpstqslni ksepvspprd rtttpsrypq htrheagrsp 421 vdslsscsss ydgsdredhr nefhspiglt rpspderesp svkrmrlseg wat
(SEQ ID NO: 18) 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
(dimerization interface), 87-134 (splicing variant), 107-134 (splicing variant), 110-156 (Holliday junction regulator protein family), 271-278 (splicing variant), and 368-399 (transcription repressor binding site).
Human MEF2C nucleotide sequence. The start and stop codons of the coding sequence are bold and underlined.
1 aagggggcaa agcctcggtc ttcatagaaa aggagaggag gcaaacgcag cccaaactgg
61 ggggtttctc ttcaaagcca gctggtctgg ctttattctg caggaatttt tttacctgtc
121 agggtttgga caacaaagcc ctcagcaggt gctgacgggt acaacttcct ggagaagcag
181 aaaggcactg gtgccaaaga agagttgcaa actgtgaagt aacttctatg aagagatgaa
241 gtaaagaacg gaaggcaaat gattgtggca gtaaagaagt gtatgtgcag gaacgaatgc
301 aggaatttgg gaactgagct gtgcaagtgc tgaagaagga gatttgtttg gaggaaacag
361 gaaagagaaa gaaaaggaag gaaaaaatac ataatttcag ggacgagaga gagaagaaaa
421 acggggacta tggggagaaa aaagattcag attacgagga ttatggatga acgtaacaga
481 caggtgacat ttacaaagag gaaatttggg ttgatgaaga aggcttatga gctgagcgtg
541 ctgtgtgact gtgagattgc gctgatcatc ttcaacagca ccaacaagct gttccagtat
601 gccagcaccg acatggacaa agtgcttctc aagtacacgg agtacaacga gccgcatgag
661 agccggacaa actcagacat cgtggaggca ttgaacaaga aagaaaacaa aggctgtgaa
721 agccccgatc ccgactcctc ttatgcactc accccacgca ctgaagaaaa atacaaaaaa
781 attaatgaag aatttgataa tatgatcaag agtcataaaa ttcctgctgt tccacctccc
841 aacttcgaga tgccagtctc catcccagtg tccagccaca acagtttggt gtacagcaac
901 cctgtcagct cactgggaaa ccccaaccta ttgccactgg ctcacccttc tctgcagagg
961 aatagtatgt ctcctggtgt aacacatcga cctccaagtg caggtaacac aggtggtctg
1021 atgggtggag acctcacgtc tggtgcaggc accagtgcag ggaacgggta tggcaatccc
1081 cgaaactcac caggtctgct ggtctcacct ggtaacttga acaagaatat gcaagcaaaa
1141 tctcctcccc caatgaattt aggaatgaat aaccgtaaac cagatctccg agttcttatt
1201 ccaccaggca gcaagaatac gatgccatca gtgaatcaaa ggataaataa ctcccagtcg
1261 gctcagtcat tggctacccc agtggtttcc gtagcaactc ctactttacc aggacaagga
1321 atgggaggat atccatcagc catttcaaca acatatggta ccgagtactc tctgagtagt
1381 gcagacctgt catctctgtc tgggtttaac accgccagcg ctcttcacct tggttcagta
1441 actggctggc aacagcaaca cctacataac atgccaccat ctgccctcag tcagttggga
1501 gcttgcacta gcactcattt atctcagagt tcaaatctct ccctgccttc tactcaaagc
1561 ctcaacatca agtcagaacc tgtttctcct cctagagacc gtaccaccac cccttcgaga
1621 tacccacaac acacgcgcca cgaggcgggg agatctcctg ttgacagctt gagcagctgt
1681 agcagttcgt acgacgggag cgaccgagag gatcaccgga acgaattcca ctcccccatt
1741 ggactcacca gaccttcgcc ggacgaaagg gaaagtccct cagtcaagcg catgcgactt
1801 tctgaaggat gggcaacatg atcagattat tacttactag tttttttttt tttcttgcag
1861 tgtgtgtgtg tgctatacct taatggggaa ggggggtcga tatgcattat atgtgccgtg
1921 tgtggaaaaa aaaaaagtca ggtactctgt tttgtaaaag tacttttaaa ttgcctcagt
1981 gatacagtat aaagataaac agaaatgctg agataagctt agcacttgag ttgtacaaca
2041 gaacacttgt acaaaataga ttttaaggct aacttctttt cactgttgtg ctcctttgca
2101 aaatgtatgt tacaatagat agtgtcatgt tgcaggttca acgttattta catgtaaata
2161 gacaaaagga aacatttgcc aaaagcggca gatctttact gaaagagaga gcagctgtta
2221 tgcaacatat agaaaaatgt atagatgctt ggacagaccc ggtaatgggt ggccattggt 2281 aaatgttagg aacacaccag gtcacctgac atcccaagaa tgctcacaaa cctgcaggca 2341 tatcattggc gtatggcact cattaaaaag gatcagagac cattaaaaga ggaccatacc 2401 tattaaaaaa aaatgtggag ttggagggct aacatattta attaaataaa taaataaatc 2461 tgggtctgca tctcttatta aataaaaata taaaaatatg tacattacat tttgcttatt 2521 ttcatataaa aggtaagaca gagtttgcaa agcatttgtg gctttttgta gtttacttaa 2581 gccaaaatgt gtttttttcc ccttgatagc ttcgctaata ttttaaacag tcctgtaaaa 2641 aaccaaaaag gactttttgt atagaaagca ctaccctaag ccatgaagaa ctccatgctt 2701 tgctaaccaa gataactgtt ttctctttgt agaagttttg tttttgaaat gtgtatttct 2761 aattatataa aatattaaga atcttttaaa aaaatctgtg aaattaacat gcttgtgtat 2821 agctttctaa tatatataat attatggtaa tagcagaagt tttgttatct taatagcggg 2881 aggggggtat atttgtgcag ttgcacattt gagtaactat tttctttctg ttttctttta 2941 ctctgcttac attttataag tttaaggtca gctgtcaaaa ggataacctg tggggttaga 3001 acatatcaca ttgcaacacc ctaaattgtt tttaatacat tagcaatcta ttgggtcaac 3061 tgacatccat tgtatatact agtttctttc atgctatttt tattttgttt tttgcatttt 3121 tatcaaatgc agggcccctt tctgatctca ccatttcacc atgcatcttg gaattcagta 3181 agtgcatatc ctaacttgcc catattctaa atcatctggt tggttttcag cctagaattt 3241 gatacgcttt ttagaaatat gcccagaata gaaaagctat gttggggcac atgtcctgca 3301 aatatggccc tagaaacaag tgatatggaa tttacttggt gaataagtta taaattccca 3361 cagaagaaaa atgtgaaaga ctgggtgcta gacaagaagg aagcaggtaa agggatagtt 3421 gctttgtcat ccgtttttaa ttattttaac tgacccttga caatcttgtc agcaatatag 3481 gactgttgaa caatcccggt gtgtcaggac ccccaaatgt cacttctgca taaagcatgt 3541 atgtcatcta ttttttcttc aataaagaga tttaatagcc atttcaagaa atcccataaa 3601 gaacctctct atgtcccttt ttttaattta aaaaaaatga ctcttgtcta atattcgtct 3661 ataagggatt aattttcaga ccctttaata agtgagtgcc ataagaaagt caatatatat 3721 tgtttaaaag atatttcagt ctaggaaaga ttttccttct cttggaatgt gaagatctgt 3781 cgattcatct ccaatcatat gcattgacat acacagcaaa gaagatatag gcagtaatat 3841 caacactgct atatcatgtg taggacattt cttatccatt ttttctcttt tacttgcata 3901 gttgctatgt gtttctcatt gtaaaaggct gccgctgggt ggcagaagcc aagagacctt 3961 attaactagg ctatattttt cttaacttga tctgaaatcc acaattagac cacaatgcac 4021 ctttggttgt atccataaag gatgctagcc tgccttgtac taatgtttta tatattaaaa 4081 aaaaaaaatc tatcaaccat ttcatatata tcccactact caaggtatcc atggaacatg 4141 aaagaataac atttatgcag aggaaaaaca aaaacatccc tgaaaatata cacactcata 4201 cacacacacg cacaggggaa taaaataaga aaatcatttt cctcaccata gacttgatcc 4261 catccttaca acccatcctt ctaacttgat gtgtataaaa tatgcaaaca tttcacaaat 4321 gttctttgtc atttcaaaat actttagtat atcaatatca gtagatacca gtgggtggga 4381 aagggtcatt acatgaaaat atgaagaaat agccatatta gttttttaac ctgcaatttg 4441 cctcagcaac aaagaaaaag tgaattttta atgctgaaga taaagtaagc taaagtacca 4501 gcagaagcct tggctattta tagcagttct gacaatagtt ttataagaac atgaagagaa 4561 cagaatcact tgaaaatgga tgccagtcat ctcttgttcc cactactgaa ttcttataaa 4621 gtggtggcaa gatagggaag ggataatctg agaattttta aaagatgatt taatgagaag 4681 aagcacaatt ttgattttga tgagtcactt tctgtaaaca atcttggtct atctttaccc 4741 ttatacctta tctgtaattt accatttatt gtatttgcaa agctagtatg gtttttaatc 4801 acagtaaatc ctttgtattc cagactttag ggcagagccc tgagggagta ttattttaca 4861 taacccgtcc tagagtaaca ttttaggcaa cattcttcat tgcaagtaaa agatccataa 4921 gtggcatttt acacggctgc gagtattgtt atatctaatc ctattttaaa agatttttgg 4981 taatatgaag cttgaatact ggtaacagtg atgcaatata cgcaagctgc acaacctgta 5041 tattgtatgc attgctgcgt ggaggctgtt tatttcaacc tttttaaaaa ttgtgttttt 5101 tagtaaaatg gcttattttt tcccaaaggt ggaatttagc attttgtaat gatgaatata 5161 aaaatacctg tcatccccag atcatttaaa agttaactaa agtgagaatg aaaaaacaaa 5221 attccaagac actttttaaa agaatgtctg ccctcacaca cttttatgga tttgtttttc 5281 ttacataccc atcttttaac ttagagatag cattttttgc cctctttatt ttgttgtttg 5341 tttctccaga gagtaaacgc tttgtagttc tttctttaaa aaacattttt tttaaagaag
5401 aagaagccac ttgaaccctc aataaaggct gttgcctaag catggcatac ttcatctgtt
5461 ctcatttgtg ccatctgccg tgatgtcgtc acttttatgg cgttaatttc ctgccactac
5521 agatcttttg aagattgctg gaatactggt gtctgttaga atgcttcaga ctacagatgt
5581 aattaaaggc ttttcttaat atgttttaac caaagatgtg gagcaatcca agccacatat
5641 cttctacatc aaatttttcc attttggtta ttttcataat ctggtattgc attttgcctt
5701 ccctgttcat acctcaaatt gattcatacc tcagtttaat tcagagaggt cagttaagtg
5761 acggattctg ttgtggtttg aatgcagtac cagtgttctc ttcgagcaaa gtagacctgg
5821 gtcactgtag gcataggact tggattgctt cagatggttt gctgtatcat ttttcttctt
5881 tttcttttcc tggggacttg tttccattaa atgagagtaa ttaaaatcgc ttgtaaatga
5941 gggcatacaa gcatttgcaa caaatattca aatagaggct cacagcggca taagctggac
6001 tttgtcgcca ctagatgaca agatgttata actaagttaa accacatctg tgtatctcaa
6061 gggacttaat tcagctgtct gtagtgaata aaagtgggaa attttcaaaa gtttctcctg
6121 ctggaaataa ggtataattt gtattttgca gacaattcag taaagttact ggctttctta 6181 gtgaaaaaaa aaaa
(SEQ ID NO: 19) GenBank Accession NM_001131005.2 (GI: 301069378), incorporated herein by reference.
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).
Binding Ligands for Biomarkers
As used herein, the term "antibody" refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e. , molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F(ab )2 fragments, and an Fab expression library. By "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 (Kd > 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. Within light and heavy chains, the 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. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form the antibody binding site.
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
In general, 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, IgG2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.
The term "antigen-binding site" or "binding portion" refers to the part of the
immunoglobulin molecule that participates in antigen binding. 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. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, 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. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term "epitope" 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 μΜ; preferably < 100 nM and most preferably < 10 nM.
Antibodies can be produced according to any method known in the art.
Methods of preparing monoclonal antibodies are known in the art. For example, monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In 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. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a full length protein or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") 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. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
In some examples 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. In some instances, 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. In general, 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. Accordingly, 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
corresponding sequence from a non-human species.
In another examples 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). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in most respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications: Marks et al. 1992.
Bio/Technology 10:779-783; Lonberg et al. 1994. Nature 368:856-859; Morrison. 1994. Nature 368:812-13; Fishwild et al. 1996. Nature Biotechnology 14:845-51; Neuberger. 1996. Nature Biotechnology 14:826; Lonberg and Huszar. 1995. Intern. Rev. Immunol. 13:65-93. U.S. Pat. No. 6,719,971 also provides guidance to methods of generating humanized antibodies.
Exemplary antibodies against human MESP1 protein include, but are not limited to, antibodies obtained from "Thermo Scientific online" (e.g., Cat. No. MA5- 15645; and more can be found at its website www.pierce-antibodies.com), antibodies obtained from "abcam.com" (e.g., ab77013, abl71427, abl29387, ab86419, and more can be found at its website
www.abcam.com), antibodies obtained from Santa Cruz Biotech (e.g., sc-163077, sc-163078, sc- 163074, sc- 163076, and more can be found at its website www.scbt.com); any commercially available antibodies against MESP1, and any antibodies that are generated by known method in the art utilizing the full-length protein or a fragment of human MESP1 (e.g., residues 83-140, residues 83-122, residues 97-140, residues 16.-167, residues 182-185, any fragment or full length of SEQ ID NO: 1).
Exemplary antibodies against HuR (ELAVL1) protein include, but are not limited to, antibodies obtained from "antibodies online" (e.g., Cat. No. ABIN577055; Cat. No.
ABIN659117; Cat. No. ABIN652911; Cat. No. ABIN396456; Cat. No. ABIN307186, and more can be found at its website www.antibodies-online.com), antibodies obtained from "abcam.com" (e.g., ab54987, abl35740, abl37471, abl36542, and more can be found at its website
www.abcam.com), antibodies obtained from Santa Cruz Biotech (e.g., sc-5261, sc-20694, sc- 374285 and more can be found at its website www.scbt.com); any commercially available antibodies against HuR, and any antibodies that are generated by known method in the art utilizing the full-length protein or a fragment of human HuR (e.g., residues 243-326, residues 180-183, residues 157-167, residues 146-156, residues 141-143, residues 107-185, residues 106- 189, residues 19-96, residues 19-326, any fragment or full length of SEQ ID NO: 14).
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).
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, 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, 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).
Amplification-Based Detection Methods
RT-PCR is in particularly broad use as a method for amplifying mRNA sequences of interest. Details regarding the use of RT-PCR, PCR, and other amplification methods can be found in any of a variety of standard texts. One of skill will appreciate that essentially any RNA can be converted into DNA suitable for PCR expansion using a reverse transcriptase and a polymerase.
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. For example, primers can be designed using any suitable software program, such as
LASERGENE®, e.g. , 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).
In various embodiments, a pair of primers that is complementary to a single transcript is used. In certain embodiments, one or more of the primers used comprises a sequence of nucleic acids that is not complementary to the transcript. For example, 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). In some embodiments, 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.
In some embodiments, 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. In some embodiments, the primers are not labelled, and the amplicons are visualized following their size resolution, e.g. , following agarose or acrylamide gel electrophoresis. In some embodiments, ethidium bromide staining of the PCR amplicons following size resolution allows visualization of the different size amplicons.
It is not intended that the primers of the invention be limited to generating an amplicon of any particular size. For example, 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. In some embodiments, amplification produces an amplicon at least 20 nucleotides in length, or alternatively, at least 50 nucleotides in length, or
alternatively, 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.
In some embodiments, PCR detection using dual-labelled fluorogenic oligonucleotide probes, commonly referred to as "TaqMan™" 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. During the extension phase of PCR, the probe is cleaved by 5' nuclease activity of the polymerase used in the reaction, thereby releasing the reporter from the oligonucleotide- quencher and producing an increase in reporter emission intensity. Accordingly, TaqMan™ 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 TaqMan™ reagents are commercially available, e.g. , from Applied Biosystems (Division Headquarters in Foster City, Calif.) as well as from a variety of specialty vendors such as Biosearch Technologies (e.g. , black hole quencher probes). Further details regarding dual-label probe strategies can be found, e.g. , in PCT International Patent Application Publication No. WO 92/02638.
Other similar methods include e.g. fluorescence resonance energy transfer between two adjacently hybridized probes, e.g. , using the "LightCycler®" format described in U.S. Pat. No. 6,174,670, issued January 16, 2001.
Array-Based Detection
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.
A variety of probe arrays have been described in the literature and can be used to detect mRNA or amplicons thereof (e.g. , obtained using RT-PCR). For example, DNA probe array chips or larger DNA probe array wafers (from which individual chips would otherwise be obtained by breaking up the wafer) 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.
In some implementations, the polynucleotide to be analyzed (e.g. , an mRNA encoding a MESP1, SCN5A, HU, and/or MEF2C) is isolated, amplified and labelled with biotin and/or a fluorescent reporter group. The labelled nucleic acid may then be incubated with the array using a fluidics station and hybridization oven. The array can be washed and or stained or counter- stained, as appropriate to the detection method. After hybridization, washing and staining, the array is inserted into a scanner, where patterns of hybridization are detected. The hybridization data are collected as light emitted from the fluorescent reporter groups already incorporated into the labelled nucleic acid, which is now bound to the probe array. Probes that most clearly match the labelled nucleic acid produce stronger signals than those that have mismatches. Since the sequence and position of each probe on the array are known, by complementarity, the identity of the nucleic acid sample applied to the probe array can be identified.
In one embodiment, 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.
In various embodiments, 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).
Examples
Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Example 1: mRNA expression of MESPl in WBCs is decreased in both SCN5A(-) patients and SCN5A(+) patients
Twenty-five control patients, 25 BrS patients without SCN5A mutations, and 20 BrS patients with SCN5A mutations were evaluated. Blood 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-Zol™ 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.
WBC levels of MESPl were significantly lower in BrS patients than in a normal control group (1.08 + 0.38 in control group versus 0.66 + 0.38 in BrS patients without SCN5A mutation, 0.44 + 0.42 in BrS patients with SCN5A mutation respectively, P=0.001 by AVON analysis). T- tests revealed that the difference between the mRNA expression of MES 1P1 in WBCs was decreased in both SCN5A(-) (P=0.012 vs. control) and SCN5A(+) (P=0.000 vs. control) groups. No difference was observed between the two BrS groups in MESPl expression (P=0.215). The area under the ROC analysis curve for prediction of BrS using MESPl levels was 0.775 (95% CI: 0.668-0.882, asymptotic Sig. =0.000). At the optimal cutoff, the corresponding maximum sensitivity and specificity were 0.62 (95% CI: 0.47-0.76) and 0.88 (0.69-0.97) respectively (and summarized in the tables below).
Area under the Receiver Operating Characteristics (ROC) prediction of BrS using MESPl levels:
Figure imgf000062_0001
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. The area under the ROC analysis curve for prediction of BrS with an SCN5A mutation using SCN5A, MEF2C and HuR mRNA levels in WBCs was 0.847 (95% CI 0.752-0.942, asymptotic Sig. =0.000), 0.685 (95% CI 0.542-0.828, asymptotic Sig. =0.016) and 0.777 (95% CI 0.652-0.902, asymptotic and HuR for SCN5A(+) BrS diagnosis was 17.5 (95% CI: 8.06-37.86), 4.9 (95% CI: 2.61-9.17) and 23.5 (95% CI: 9.39- 8,80), respectively (and summarized in the tables below).
Area under the DOR (diagnostics odds ratio) analysis for BrS diagnosis (at the optimal cut off, with SCN5A mutation):
Figure imgf000062_0002
^Circulating MES Ί is useful as a biomarker
Area under the ROC analysis (prediction of BrS with an SCN5A mutation) in WBCs: Index Test mRNA levels in WBCs (95% CD Asvmptotic Sig.
SCN5A mutation 0.847 (0.752-0.942) 0.000
MEF2C 0.685 (0.542-0.828 0.016
HuR 0.777 (0.652-0.902) 0.000
Area under the Receiver Operating Characteristics (ROC) prediction of BrS using MESPl levels:
Figure imgf000063_0001
The results indicated that assessment of circulating MESPl is useful as a biomarker for BrS diagnosis, while decreased SCN5A, MEF2C and HuR mRNA in WBCs is associated with BrS patients with an SCN5A mutation. The results also indicated that decreased expression of SCN5A, MEF2C, MESPl, and HuR may be pathophysiologically related to BrS.
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. OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference.
Genbank, NCBI, UniProt, or other submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the disclosure encompassed by the appended claims.

Claims

CLAIMS What is claimed is:
1. A method for diagnosing Brugada Syndrome (BrS) comprising,
providing a test sample from a subject, wherein said sample comprises a circulating cell or a bodily fluid,
performing a reaction in vitro to yield a complex comprising a mesoderm posterior protein 1 (MESPl) protein or nucleic acid and a binding agent, and
detecting said complex, wherein a decrease in the level of the complex compared to a normal control is indicative of BrS.
2. The method of claim 1, further comprising computing a level of said MESPl protein or MESPl nucleic acid, wherein least 30% decrease in the level of said protein or nucleic acid compared to a normal control is indicative of BrS.
3. The method of claim 1, further comprising computing a level of a sodium channel, voltage gated, type V alpha (SCN5A) protein, an HU protein, or a myocyte enhancer factor-2 (MEF2C) protein with a binding agent, wherein a decrease in the level of said compared to a normal control is indicative of BrS.
4. The method of claim 1, further comprising computing a level of an SCN5A variant, wherein an increase in the level of the variant compared to a normal control is indicative of BrS.
5. The method of claim 1, wherein the binding agent comprises an antibody or a fragment thereof, a detectable protein, or a fragment thereof, or a nucleic acid molecule.
6. The method of claim 1, wherein the in vitro reaction comprises quantitative PCR or Northern Blot.
7. The method of claim 1, wherein the nucleic acid comprises cDNA.
8. The method of claim 1, wherein said MESP1 binding agent comprises an antibody.
9. The method of claim 8, wherein the antibody is conjugated to a detectable moiety.
10. The method of claim 8, wherein said antibody comprises a polyclonal antibody or a monoclonal antibody.
11. The method of claim 1, wherein said binding agent is attached to a solid support.
12. The method of claim 11, wherein said solid support comprises a strip, a polymer, a bead, or a nanoparticle.
13. The method of claim 9, wherein the detectable moiety comprises a fluorescent marker selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine, and 152 Eu; wherein the
125 detectable moiety comprises a radioactive agent selected from the group consisting of I, tritium, 75-selenomethonine, and 64-copper; wherein the detectable moiety comprises a chemiluminescent compound selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
14. The method of claim 4, wherein said SCN5A variant comprises an amino acid sequence of human SCN5A comprising SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 11, or a nucleic acid sequence of human SCN5A comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
15. The method of claim 3, wherein said SCN5A binding agent comprises an antibody.
16. The method of claim 15, wherein the antibody is conjugated to a detectable moiety.
17. The method of claim 15, wherein said antibody comprises a polyclonal antibody or a monoclonal antibody.
18. The method of claim 3, wherein said binding agent is attached to a solid support.
19. The method of claim 18, wherein said solid support comprises a strip, a polymer, a bead, or a nanoparticle.
20. The method of claim 16, wherein the detectable moiety comprises a fluorescent marker selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine, and 152 Eu; wherein the
125 detectable moiety comprises a radioactive agent selected from the group consisting of I, tritium, 75-selenomethonine, and 64-copper; wherein the detectable moiety comprises a chemiluminescent compound selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
21. The method of claim 3, wherein said HU protein or HU nucleic acid comprises an amino acid sequence or nucleic acid sequence of human HuR (SEQ ID NO: 14 and SEQ ID NO: 15, respectively) or HuB (SEQ ID NO: 16 and SEQ ID NO: 17, respectively), or a fragment thereof.
22. The method of claim 3, wherein said HU protein or HU nucleic acid comprises an amino acid sequence or nucleic acid sequence of human HuR (SEQ ID NO: 14 and SEQ ID NO: 15, respectively), or a fragment thereof.
23. The method of claim 3, wherein said HU binding agent comprises an antibody.
24. The method of claim 23, wherein the antibody is conjugated to a detectable moiety.
25. The method of claim 23, wherein said antibody comprises a polyclonal antibody or a monoclonal antibody.
26. The method of claim 3, wherein said binding agent is attached to a solid support.
27. The method of claim 26, wherein said solid support comprises a strip, a polymer, a bead, or a nanoparticle.
28. The method of claim 24, wherein the detectable moiety comprises a fluorescent marker selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine, and 152 Eu; wherein the
125 detectable moiety comprises a radioactive agent selected from the group consisting of I, tritium, 75-selenomethonine, and 64-copper; wherein the detectable moiety comprises a chemiluminescent compound selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
29. The method of claim 3, wherein said MEF2C protein or MEF2C nucleic acid comprises an amino acid sequence or nucleic acid sequence of human MEF2C (SEQ ID NO: 18 and SEQ ID NO: 19, respectively), or a fragment thereof.
30. The method of claim 3, wherein said MEF2C binding agent comprises an antibody.
31. The method of claim 30, wherein the antibody is conjugated to a detectable moiety.
32. The method of claim 30, wherein said antibody comprises a polyclonal antibody or a monoclonal antibody.
33. The method of claim 3, wherein said binding agent is attached to a solid support.
34. The method of claim 33, wherein said solid support comprises a strip, a polymer, a bead, or a nanoparticle.
35. The method of claim 31, wherein the detectable moiety comprises a fluorescent marker selected from the group consisting of fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, p-phthaldehyde and fluorescamine, and 152 Eu; wherein the
125 detectable moiety comprises a radioactive agent selected from the group consisting of I, tritium, 75-selenomethonine, and 64-copper; wherein the detectable moiety comprises a chemiluminescent compound selected from the group consisting of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
36. The method of claim 1, wherein said subject is a human.
37. The method of claim 1, further comprising contacting said protein with a stabilizing compound to yield a durable protein/mRNA complex.
38. The method of claim 1, wherein said test sample comprises plasma, whole blood, serum, saliva or urine.
39. The method of claim 1, wherein said test sample comprises serum.
40. The method of claim 1, wherein the level of said protein is decreased by at least 10% compared to said control.
41. The method of claim 1, wherein said test sample comprises a population of enriched white blood cells.
42. The method of claim 41, wherein said population comprises a buffy coat fraction of total white blood cells.
43. The method of claim 1, wherein said test sample comprises a monocyte.
44. The method of claim 1, wherein said circulating cell comprises a T-cell.
45. The method of claim 1, wherein said cell is lysed prior to said contacting step.
46. The method of claim 1, wherein said detecting step comprises an ELISA (enzyme-linked immunosorbent) assay, a Western blot, a mass spectrometry, a radioimmunoassay, and a fluoroimmunoas s ay .
47. A kit comprising an agent for detecting level of an MESP1 protein for diagnosing Brugada Syndrome,
wherein the agent binds to an MESP1 protein or nucleic acid encoding said protein, and yields a complex comprising said MESP1 protein or nucleic acid and said binding agent
and instructions for using the agent for diagnosing Brugada Syndrome.
48. The kit of claim 47 further comprising reagents used to detect said complex.
49. The kit of claim 47, further wherein said agent is attached to a solid support.
50. The kit of claim 49, wherein said solid support is a test strip.
51. A method of treating Brugada Syndrome in a subject comprising,
measuring the level of an MESP1 protein in a subject, wherein the decreased level of an MESP1 protein indicates Brugada Syndrome, and
administering to said subject, an active agent for treating BrS.
52. The method of claim 51, wherein said active agent comprises 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.
53. The method of claim 52, wherein the antiarrhythmia agent comprises a Singh Vaughan Williams (SVW) Class I, IA, IB, IC, II, III, IV, or V, IC, a fast-channel blocker, a beta blocker, a slow channel blocker, a sodium channel blocking agent, a potassium channel blocking agent, a calcium channel blocking agent, 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, or Magnesium Sulfate, Encainide, NAD+ or mitoTEMPO.
54. The method of claim 51, further comprising repeating said providing, performing, and detecting steps, wherein the repeating is at least once before treatment and at least once after treatment, to evaluate or monitor a response to treatment, wherein a change in the level of said protein and/or said nucleic acid indicates a response to said treatment.
55. A diagnostic test system comprising,
obtaining test results data representing levels of a marker in at least one biological sample,
collecting test results data,
a means for computing an index value from said marker, wherein the index value comprises a Brugada Syndrome risk score, and
a means of reporting the index value.
56. The diagnostic test system of claim 55, wherein the marker comprises an an MESP1 protein, an HU protein, an SCN5A protein or variant, or an MEF2C protein.
57. The diagnostic test system of claim 55, wherein the index value uses said marker measurement data, and
wherein the index score is correlated with risk of developing Brugada Syndrome.
58. The diagnostic test system of claim 55, wherein the biological sample comprises plasma, whole blood, serum, saliva or urine.
59. A diagnostic device comprising a solid support and an MESP1 binding agent immobilized on said support.
70
RECTIFIED (RULE 91) - ISA/US
60. A diagnostic device comprising a plurality of immobilized binding agents, wherein a first binding agent binds to HuR or HuB, a second binding agent binds to SCN5A, a third binding agent binds to MESPl, or a fourth binding agent binds to MEF2C.
61. A complex comprising (i) an isolated nucleic acid having at least 90% sequence identity to the sequence of a mesoderm posterior basic helix-loop-helix transcription factor 1 (MESPl) transcript, hybridized to (ii) an isolated nucleic acid that is conjugated to a biological or a chemical probe.
62. A complex comprising (i) an isolated nucleic acid having at least 90% sequence identity with a complementary DNA (cDNA) of MESPl, hybridized to (ii) an isolated nucleic acid that is conjugated to a biological or a chemical probe.
63. A method of determining risk of Brugada Syndrome of a subject, the method comprising:
(i) providing RNA from white blood cell of said subject;
(ii) reverse transcribing the isolated RNA to generate cDNA of an MESPl gene;
(iii) amplifying said cDNA with primers that are at least about 90%, 95%, 96%, 97%, 98%, 99% complementary to SEQ ID NO: 2 or that are 100% complementary to SEQ ID NO: 2, wherein the primers are conjugated to a biological or a chemical probe; and
(iv) detecting a signal from the probe, thereby detecting expression level of MESPl;
wherein the expression level of MESPl determines risk of Brugada Syndrome of the subject.
64. The method of claim 63, wherein the subject is diagnosed with Brugada Syndrome.
65. The method of claim 63, wherein the detecting the signal further comprises determining whether the signal is increased or decreased relative to a control, wherein decreased level of the signal compared to the control determines arrhythmic risk of the subject.
71
RECTIFIED (RULE 91) - ISA/US
66. The method of one of claims 63-65, wherein the subject does or does not carry mutations in the sodium channel, voltage gated, type V alpha subunit (SCN5A).
67. The method of claim 66, wherein the level of the signal in the subject that does carry SCN5A mutations is not increased or decreased compared to the subject that does not carry SCN5A mutations.
68. A method of diagnosing risk of heart failure in a subject, the method comprising:
(i) providing RNA from white blood cell of said subject;
(ii) reverse transcribing the isolated RNA to generate cDNA of MESP1 gene;
(iii) primers that are at least about 90%, 95%, 96%, 97%, 98%, 99%
complementary to SEQ ID NO: 2 or that are 100% complementary to SEQ ID NO: 2, wherein the primers are conjugated to a biological or a chemical probe; and
(iv) detecting a signal from the probe, thereby detecting expression level of MESP1;
wherein the expression level of MESP1 diagnoses the risk of heart failure in the subject.
69. The method of claim 68, wherein the subject is diagnosed with Brugada Syndrome.
70. The method of claim 68, wherein the expression level of MESP1 predicts arrhythmic risk of the subject.
71. The method of claim 70, wherein the detecting the signal further comprises determining whether the signal is increased or decreased relative to a control, wherein decreased level of the signal compared to the control predicts that the subject is at risk of class C or class D heart failure.
72
RECTIFIED (RULE 91) - ISA/US
72. The method of one of claims 68-71, wherein the subject does or does not carry mutations in the sodium channel, voltage gated, type V alpha subunit (SCN5A).
73. The method of claim 72, wherein the level of the signal in the subject that does carry SCN5A mutations is not increased or decreased compared to the subject that does not carry SCN5A mutations.
74. A method of treating a subject at risk of class C or class D heart failure, or at arrhythmic risk, the method comprising determining expression level of MESPl in the white blood cell of said subject; wherein decreased level of MESPl expression compared to a control selects said subject as a candidate for said treatment; and administering a therapeutic agent to said subject, thereby treating class C or class D heart failure, or arrhythmic risk in said subject.
75. The method of claim 74, wherein the expression level of MESPl is determined by a method comprising: providing RNA from white blood cell of said subject;
(i) reverse transcribing the isolated RNA to generate cDNA of MESPl gene;
(ii) amplifying said cDNA with primers that are at least about 90%, 95%, 96%, 97%, 98%, 99% complementary to SEQ ID NO: 2 or that are 100% complementary to SEQ ID NO: 2, wherein the primers are conjugated to a biological or a chemical probe; and
(iii) detecting a signal from the probe, thereby determining the expression level of MESPl.
76. A kit comprising nucleic acids that are at least about 80, 85, 90%, 95%, 96%, 97%, 98%, 99% complementary to SEQ ID NO: 2, or that are 100% complementary to SEQ ID NO: 2, over the entire lengths thereof.
77. An antibody for use in diagnosing Brugada Syndrome (BrS), wherein said diagnosing comprises the steps of:
providing a test sample from a subject, wherein said sample comprises a circulating cell or a bodily fluid,
73
RECTIFIED (RULE 91) - ISA/US performing a reaction in vitro to yield a complex comprising a mesoderm posterior protein 1 (MESPl) protein or nucleic acid and a binding agent, and
detecting said complex, wherein a decrease in the level of the complex compared to a normal control is indicative of BrS.
78. The antibody for use according to claim 77, wherein said antibody comprises an antibody against MESPl.
79. The antibody for use according to claim 78, wherein said antibody further comprises an antibody against an HU protein, SCN5A, and/or MEF2C.
80. A method for diagnosing Brugada 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 mesoderm posterior protein 1 (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.
81. The method of claim 80, wherein 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.
82. A method for treating Brugada Syndrome in a subject who has been diagnosed with Brugada Syndrome according to the method claim 80, comprising implanting a cardioverter- defibrillator (ICD) into the subject.
83. A method for identifying whether a subject who has Brugada Syndrome is at risk of suffering from suddent cardiac death (SCD), comprising
74
RECTIFIED (RULE 91) - ISA/US (a) providing a test sample from said subject, wherein said test sample comprises a circulating cell or a bodily fluid;
(b) assaying the level of a mesoderm posterior protein 1 (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.
84. The method of claim 83, wherein 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.
85. A method for treating Brugada Syndrome in a subject who has been identified as at risk of dying from SCD according to the method claim 83, comprising implanting a cardioverter- defibrillator (ICD) into the subject.
86. A method for monitoring the risk of sudden cardiac death (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
75
RECTIFIED (RULE 91) - ISA/US (b) assaying the level of the MESPl protein or MESPl protein-encoding mRNA in the test sample.
87. A method for identifying whether a subject who comprises Brugada Syndrome is at risk of dying from sudden cardiac death (SCD), comprising
(a) providing a test sample from said 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) (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.
88. A method for treating Brugada Syndrome in a subject who has been identified as at risk of dying from SCD according to the method claim 87&8, comprising implanting a cardioverter- defibrillator (ICD) into the subject.
89. A method for identifying whether a therapy has improved Brugada Syndrome in a subject, comprising
(a) providing a pre-therapy test sample from said subject;
(b) assaying a pre-therapy level of a MESPl protein or MESPl 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 MESPl protein or MESPl protein-encoding mRNA in the post-therapy test sample; and
76
RECTIFIED (RULE 91) - ISA/US (f) identifying the therapy as having improved Brugada Syndrome in the subject if the pre-therapy level of the MESPl protein or MESPl protein-encoding mRNA is lower than the post-therapy level of the MESPl protein or MESPl protein-encoding mRNA.
77
RECTIFIED (RULE 91) - ISA/US
PCT/US2016/047596 2015-08-20 2016-08-18 Circulating biomarker for brugada syndrome WO2017031342A1 (en)

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