WO2012154452A1 - Stratification, therapies, targeted treatment and prevention of life-threatening ventricular tachya - Google Patents

Stratification, therapies, targeted treatment and prevention of life-threatening ventricular tachya Download PDF

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WO2012154452A1
WO2012154452A1 PCT/US2012/036004 US2012036004W WO2012154452A1 WO 2012154452 A1 WO2012154452 A1 WO 2012154452A1 US 2012036004 W US2012036004 W US 2012036004W WO 2012154452 A1 WO2012154452 A1 WO 2012154452A1
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mutations
syndrome
long
patients
channel
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PCT/US2012/036004
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French (fr)
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Arthur J. Moss
Ilan Goldenberg
Jin OUCHI
Coeli Maria BASTOS LOPES
Alon Eli Bar SHESHET
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University Of Rochester
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates generally to systems and methods for risk stratification, therapies, targeted treatments and prevention of cardiac disorders, and more specifically to Systems And Methods For Risk Stratification, Therapies, Targeted Treatment And Prevention of Life- Threatening Ventricular Tachyarrhythmias And Sudden Cardiac- Death For Patients With Congenital Type- 1 Long QT Syndrome (LQT1 ),
  • LQTS Congenital long-QT syndromes
  • LQTS type- 3 LQT
  • Patients with LQTl have increased risk for life-threatening ventricular tachyarrhythmias and sudden cardiac death during sympathetic activation, associated predominantly with exercise activity or swimming. Accordingly, current guidelines recommend non-specific treatment wit beta-blocker therapy in. LQTS patients. Despite this, however, there are still a considerable number of LQTl patients who experience life-threatening ventricular tachyarrhythmias during beta-blocker therapy. Furthermore, despite multiple attempts for risk stratification studies in long QT syndrome, currently there is limited ability to identify LQTS patients who will not respond to beta-blocker therapy .
  • C-loop mutations can be used to distinguish between LQTl women with a high or low risk of sudden death.
  • Systems And Methods for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden 5 cardiac death tor patients with congenital type-1 long QT Syndrome comprising an instrument for genetic testing of LQTl syndrome and a treatment construct using data from genetic testing for therapy of LQTl syndrome.
  • Figure I is a diagram depicting frequency and location of mutations in the CNQ1 Potassium Channel from a study of 860 subjects;
  • Figure 2 is a graph of Kaplan-Meier estimates of cumulative probability of a life threatening cardiac event by mutation location and type;
  • Figure 3 is a bar graph depicting risk of life threatening cardiac events by mutation location, and beta blocker treatment;
  • Figure 4 depicts regulation of LOT.! mutant channels by PKA;
  • Figure 5 is a diagram depicting risk stratification for A.CA or SCD in LQTS patients
  • Figure 6 is a diagram depicting risk, stratification for ACA or SCD in LQl ' l patients according t the presen i invention ;
  • Figure 7 is a diagram depicting methods of targeted treatment of the present invention.
  • Figure 8 is a diagram depicting data sources used for treatment, constructs of the present invention.
  • Figure 9 is a diagram depicting a method of determining LQT1 patient responders and non- responders to beta blocker therapy:
  • Figure 10 is a diagram depicting a method of determining LQTI patients at Increased or lower risk of exercise triggered events.
  • Figure 1 .1 is a diagram depicting a .method of determining, female LQT.I patients at increased risk of sudden cardiac death.
  • the present invention will be described in connection with a preferred embodiment, however, it will be understood that there is no intent to limit the invention to the embodiment described. On the contrary, the intent is to cover all alternatives, modifications, and ' equivalents as may be included within the spirit and scope of the invention as defined by this specification, claims and the attached drawings.
  • Congenital long QT syndrome is characterized by way of an electrocardiograph (ECG) where there is a prolonged QT interval manifested by ventricular arrhythmias that may result in life -threatening cardiac events and sudden cardiac death.
  • ECG electrocardiograph
  • Current treatment for congenital long QT syndrome includes beta-blockers, implantable cardioverter-defibrillators fTCDs), and surgical left cervicothoracie sympathetic denervation (LCSD). While beta-blocker therapy is considered a first line of therapy, and some practitioners admimster beta-blockers to all long QT syndrome patients, there i s a subset of individuals with long QT syndrome genotypes where beta-blocker therapy is not effective and they remain symptomatic. There remains a need for additional long QT syndrome therapies that may require a more targeted approach to treatment.
  • LQTl Long QT syndrome type-l
  • KCNOJ KCNOJ gene encoding the a suimo.it of the slowly activating potassium channel ⁇ ».
  • Exercise is the main trigger for cardiac arrhy thmic events in patients with LQTl (Schwartz Pi, Priori SG, Spazzolini C.
  • ⁇ -adrenergic receptors Activation of ⁇ -adrenergic receptors ( ⁇ -AR) is the major signaling pathway contributing to increase in heart rate and cardiac output during exercise.
  • ⁇ -AR activation leads to activation of protein kinase A (P A). which directly phosphorylates the KCNQi suhunit, increasing 1K s function (Walsh KB, Kass RS. Regulation of a heart potassium channel b protein kinase A. and C Science. 1988;242:67-69.
  • Marx SO KLurokawa J, Reifcen S, Motoike H, D'Armiento J, Marks AR, Kass RS. Requirement of a macromoiecular signaling complex for beta adrenergic receptor modulation of the KCNQI -KCNE l potassium channel. Science. 2002;295:496-499)
  • the primary translated protein (isoform 1) of KCNQI consists of 676 amino acid residue with an intracellular N-terminus region, 6 membrane-spanning segments with two connecting cytoplasmic loops (C-loops) and an intracellular C-terniinus region (Jespersen T, Grunnet M, Olesen SP, The KCNQi potassium channel: From gene to physiological function. Physiology (B ihesdah 2005;20:408-416). Prior genotype-phenotype studies have provided important information regarding the effect of location and coding type of the channel mutations on the phenotypic manifestations and clinical course of LQTl patients.
  • the study comprised 860 patients with genetically confirmed KCNQ1 mutations derived from 170 proband -identified families.
  • the proband in each family had QTc prolongation not due to a known secondary cause.
  • the subjects were drawn from the Rochester (a ⁇ 637), the Netherlands (n ::: 94), the Japanese (n :::: 82), the Danish (n ::: 43), and the Swedish (n ::: 4) portions of the Multieenter Mutation Registry. All .subjects or their guardians provided informed consent for the genetic and clinical studies,. Patients with congenital deafness or patients with multiple LQTS associated mutations were excluded from the study. Phenotype characterization
  • Routine clinical and electrocardiographic parameters were acquired at the time of enrollment in each of the registries.
  • follow-up was censored at age 41 years to minimize the influence of coronary disease on cardiac events.
  • Measured parameters on the first recorded ECG included
  • the KCNQl mutations were identified with the use of standard genetic tests performed in academic molecular-genetic laboratories. Genetic alterations of the amino acid sequence were characterized by location and by the specific type of mutation (m!ssense, splice site, in-frame insertions/deletions, nonsense, stop eodon, and fratneshiit).
  • the membrane spanning region of the KCNQl -encoded channel was defined as the coding sequence involving amino add residues between 120-170 (SI-S2), 196-241 (S3-S4), and 263- 355 (S5-S6), with the C-loops region between residues 1 71-195 (S2-S3) and 242-262 (S4-S5), as depicted in Figure I .
  • the N-terminus region was defined before residue 120 and the C- terminus region after residue 355.
  • Figure 1 is a diagram depicting -frequency and locaiion of mutations in the KCNQl Potassium Channel from a study of 860 subjects.
  • the a subunit involves the ' -terminus (N), 6 membrane-spanning segments, 2 intraeytoplasmie loops (S2-S3 and S4-S5) and the C-temiinus portion (C).
  • N ' -terminus
  • S2-S3 and S4-S5 2 intraeytoplasmie loops
  • C-temiinus portion C.
  • the size of the circles in Figure 1 reflect the number of subjects with mutations at the respective locations.
  • Mutant KCNQl cDNA was iransfeeted in combination with WT-KCNQ 1 to mimic the heterozygous nature of the disease (WT-KCNQl :mutant KCNQl :KCN B 1-0.5:0.5:1).
  • WT-KCNQl :mutant KCNQl :KCN B 1-0.5:0.5:1 We measured ion channel currents, in the presence and absence of f orskoim, a protein kinase A activator. Details of the molecular biology and electrophysiological methods are described further later in this specification. End point
  • the primary end point of the study was ⁇ he occurrence of a first life-threatening cardiac event, comprising aborted cardiac arrest (ACA) requiring external defibrillation as part of the 5 resuscitation) or LQTS-related sudden cardiac death (SCD) abrupt in onset without evident cause, if witnessed, or death that, was not explained by any other cause if it occurred in a non- witnessed setting).
  • ACA aborted cardiac arrest
  • SCD LQTS-related sudden cardiac death
  • the probability of a first life-threatening cardiac event by the mutation-location and type subgroups was graphically displayed according to the method of Kaplan and Meier, with comparison of cumulative probability of events by the log-rank test.
  • the Cox proportional- hazards sun'ivorship model was used to evaiisate the independent contribution of clinical, and genetic factors to the first occurrence of a Hfe-ihreatening cardiac event, from birth through age 0 40 years.
  • C-loop-missense mutations was determined by adding "time-dependent ⁇ -bloeker ⁇ -by- ⁇ mutation category" interaction term to the multivariate Cox model Since almost all subjects were first- and second-degree relatives of probands, we adjusted for the effect of potential lack of independence between subjects using the robust sandwich estimator for family membership.
  • missense mutations were further categorized according to their location: there were 29 different, mutations in C-temiinus or N- terrninas regions (27 in C ⁇ terminus), 34 mutations in membrane spanning regions, and 16 mutaiions in the C-l op regions (8 in S4-S5 loop and 8 in S2-S3 loop).
  • KCNQ1 :KCNE1 DNA was expressed either at a ratio of 1 :1 or 0.5:1 in order to mimic the hapioinsuffieient phenolype in HEK293T cells.
  • Each mutant KCNQ.I was expressed in combination with WT-KCNQJ t mimic heterozygous mutation (WT-KCNQl :mutant KCNQl;K£NEI : ⁇ Q.5:Q.5: l ⁇ .
  • Each mutant current was compared to the current observed from hapioinsuffieient control channel. (0.5 ng ⁇ -KCNQI: I ng KCNE1) from the same passage cells.
  • Cells were also co-iransiected with 0.2 ng pEGFP-Nl (Clontech, La Jolia, CA) for positive identification of transfected cells by fluorescence. All the transfecfion was done by using Fugene HD trasnfection kit (Roche, Mannheim, Germany). Cells were re-plated on small glass cover slide (VW1L West Chester, PA) coated with 0.02 % gelatin 24 hours after transfectkm by using Accutase (Innovative Ceil technologies, inc., San Diego, CA) and use For experiment 48 hours after transfection.
  • Figure 2 is a graph of Kaplan-Meier estimates of cumulative probability of a life threatening cardiac event b mutation, location and type in 4 subgroups. There was a significantly higher event rate in the C- loop- mi ssense subgroup as compared with the other 3 subgroups (p log rank ⁇ 0.001). Thus, at age 40 years the rate of life threatening cardiac events was 33 percent in patients with C-loop-missense mutations as compared with ⁇ 16 percent in patients with other mutations, as illustrated in Figure 2, In Figure 2, AC ⁇ aborted cardiac arrest. LQTS ⁇ long QT syndrome. The numbers in parentheses in Figure 2 reflect the cumulative event rate at that point in time.
  • Multivariate analysis showed a significant differential effect of ⁇ -blocker therap on the outcome of patients with C-loop-missense mutation as compared with those who had other mutations, as summarized in Table 3 below.
  • S-bloeker therapy was associated with a significant 88% reduction (P :;:: 0.02) in the risk of life threatening events among patients with C-loop- missense mutations, whereas the benefit of ⁇ -blocker therapy was significantly attenuated among patients with other .mutations in the KCNQ1 channel (adjusted hazard ratio of 0.82 [P :::; 0.68j; P-value for treatinent-by-mutatioii-IocationAype interactionTM 0.04).
  • Figure 3 is a bar graph depicting risk of hie threatening cardiac events by mutation location and beta blocker treatment. Consistent with those findings, the rate of A borted Cardiac Arrest or Sudden Cardiac Death, as depicted in Figure 3, was lowest among patients with C-Ioop-missense mutations who were treated with Beta-blockers and highest among patients with C-l.oop-missen.se mutations who were not treated with beta-blockers (0,17 vs. 1.1 1 per 100 patient -years, respectively), whereas patients with other mutations in the CNQ1 channel exhibited intermediate and similar rates of 3 ⁇ 4 -threatening events with- and without beta-blocker therapy (0.36 and 0.38 per 100 patient -years, respectively.
  • Event rates per 100 person-years were calculated by dividing the number of events during the period of ⁇ -hiockcr therapy or the absence of ⁇ - b!ocker therapy by person-years, and multiplying the results by 100.
  • ACATM aborted cardiac death.
  • Table 3. M ultivariate .analysis: response to ⁇ -bloeker therapy
  • G l 68R and S225L are missense mutations located in the membrane spanning region: R243C and V254M are missense mutations located in the cytoplasmic loops region.
  • beta- blockers were associated with a significant greater reduction in the risk of life-threatening cardiac events among patients with mutations located in the C- loops as compared with all other mutations. It is conceivable that during ⁇ -adrenergic stimulation patients with mutations located in the C- loops have an unopposed increase in inward C ⁇ currents and prolongation of repolarization due to blunted P A- ediated activation of JK S . ⁇
  • 3- blockers may decrease these unopposed inward Ca ⁇ ! currents, shorten repolarization and reduce the risk for ventricular arrhythmias (Huffaker R, Lam ST, Weiss JN, ogan B. Intracellular calcium, cycling, early afierdepolarixations, and reentry in simulated long QT syndrome. Heart Rhythm. 2004; 1 :44 ⁇ - 448), whereas patients with other mutations do not exhibit such an effect.
  • the international LQTS Registry records therapies that are prescribed at the discretion of the treating physicians to enrolled subjects, and therefore ⁇ -blocker administration, was not randomized. We have therefore carried out multivariate analyses that used beta -blocker therapy as a time-dependent covariate, and have further evaluated the benefit of medical therapy with
  • beta-blockers within higher- and lower- risk subsets of LQTl patients.
  • Mutaiioii-specific information should be assessed and incorporated into the risk assessment and the management of LQT1 patients.
  • Our results highlight the importance of understanding the molecular determinants and mechanisms 5 underlying arrhythmogenesi to identify cardiac risk factors for LQ ' T! patients, and have implications for risk stratification and treatment in LOT patients.
  • Genetic testing includes all forms of testing used to test for and detect genetic disorders. Many forms of genetic testing involve examination of the DMA molecule itself, or may involve biochemical tests for enzym.es, chromosom.es, proteins, or other ] o substances. Genetic testing includes techniques to examine genes or markers near the genes.
  • Genetic testing is often times performed on a sample collected from a patient, such as a sample of blood, hair, skin, or other tissue.
  • a sample collected from a patient such as a sample of blood, hair, skin, or other tissue.
  • collection is that of a buccal smear, where a sample of ceils is collected from, the inside surface of a patient's cheek by way of a swab or brush,
  • Figure 5 is a diagram depicting risk stratification for Aborted Cardiac
  • FIG. 5 is a diagram depicting risk stratification for Aborted Cardiac Arrest or Sudden. Cardiac Death in LOT! patients according to the present invention. Mutation specific risk factors are considered in the risk stratification diagram of Figure 6.
  • steps to be taken may include, for example, performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indicati ve of long QT syndrome and processing the results of the genetic testing on a computer, identifying by way of genetic testing mutations in K.CNQJ channel ( " -loops and processing the results of 0 the genetic testing on a computer, evaluating electrocardiogram patient, information on a computer, evaluating patient clinical history on a computer; and defining a therapeutic action to be taken.
  • Therapeutic- actions may include, but are not limited to, use of a beta blocker, use of an implantable cardioverter-defibrillator, alteration of the operation of an implantable cardioverter-defibrillator (such as, for example, by programming), use of surgical left 5 cervieothoracic sympathetic denervation, or use of pharmacological agents, for example, IKs openers, PKC inhibitors, peptides based on the sequence of the c-loop region, c-loop interacting partners that may restore normal channel regulation, drugs thai mimic c-loop peptide action, drugs that bind to c-loop peptides, and drugs that bind to c-loop peptide binding partners.
  • pharmacological agents for example, IKs openers, PKC inhibitors, peptides based on the sequence of the c-loop region, c-loop interacting partners that may restore normal channel regulation, drugs thai mimic c-loop peptid
  • a system for identify ing and reducing the risk of life-threatening 0 ventricular tachyarrhythmias and sudden cardiac death in patients with congenital iype-1 long QT Syndrome comprisin an instrument for genetic testing of LQT ' l syndrome and for identifying K.C QJ channel mutations; an instrument for identifying C-loop missense mutations in the K.CNQ1 channel mutations; and a computer lor associating a therapeutic action with identified C-Ioop missense mutations in the KCNQ! channel
  • the -computer may contain listings of therapeutic actions to be taken, as will be further disclosed herein.
  • Therapeutic actions may include, but are not limited to, formulating a pharmacological agent containing a beta blocker, formulating or directing mixing or adding a type of beta blocker to a pharmacological agent, formulation of a pharmacological agent that includes, for example, IKs openers, PKC inhibitors, peptides ba.sed on the sequence of the e ⁇ !oop region, c-loop interacting partners that may restore normal channel regulation, drags that mimic c-loop peptide action, drugs that bind to c-loop peptides, or drugs that bind to c-loop peptide binding partners, use of an impS.anta.ble cardioverter-dellbrillator, programming or .modification of an implantable cardioverter-defibrillator, the use of surgical left eervicothoracie sympathetic denervation, or the like.
  • a pharmacological agent that includes, for example, IKs openers, PKC inhibitors, peptides ba.sed
  • Such a system that correlates the heretofore described genetic testing with, a therapeutic action may operate on a computer such as a laptop computer, a tablet computer, a smart phone, an instrument containing a processor, a wearable device such as a vvrisiwaieh, heart rate monitor, blood pressure monitor, or the like.
  • Therapeutic actions may also comprise alerts from the computer that are activated upon reaching a specified bodily indicator such as a specified heart rate, a specified heart rhythm, a specified pet-cent oxygen saturation in blood, a specified blood pressure, a specified chemical marker in blood, or the like.
  • the term specified is defined to be any value, numerical or otherwise, that is entered or otherwise programmed into the computer, either by a human or through another source, such, as another computer, a database, a calculation or calculations, and the like.
  • FIG 7 there i shown a diagram depicting methods of targeted risk ⁇ stratification and treatment using the present invention, instruments such as processes, techniques and equipment heretofore described for genetic testing and subsequent iden ification of mutations in C -loops of the KCNQ1 channel as show In 701 of Figure 7.
  • ECO findings are also evaluated in step 703.
  • Clinical history is also used in step 705, with the useful output being the definitio and subsequent taking of therapeutic actions In step 707 through treatment construct.
  • Treatment constructs and related therapeutic actions may include, but are not limited to, the use of beta-blockers, the use of an implantable cardioverter-defibrillator (KID), the use of surgical left cervieothoracic sympathetic denervation (LCSD).
  • KID implantable cardioverter-defibrillator
  • LCSD surgical left cervieothoracic sympathetic denervation
  • Additional possible novel therapeutic modalities (which are currently not given in LQTS patients) that, may be developed and/or evaluated as a result of the present invention include the use of various pharmacological agents such as an IKs opener (for example, 1.-364373 from.
  • an IKs opener for example, 1.-364373 from.
  • a PKC inhibitor for example, Roboxistaurin, a calcium dependent P C inhibitor
  • peptides based on the sequence of the c- ioop region or c-ioop interacting partners such as KCNQ1(S6)/ .CNQ 1 (Cterm /KCNEi(C- terra) that may restore normal channel regulation, drugs tha mimic e-loop peptide action such as drugs that can. either bind to c-loop peptides or to c-ioop peptide binding partners, or the like.
  • Figure 8 is a diagram depicting data sources used for treatment constructs of the present invention.
  • the data sources may, in one embodiment of the present invention, be contained in a data storage device such as a hard disk, solid state memory, an optical device, a magnetic device, a semiconductor device, or the like.
  • the data sources contained on physical media may also be organized or patterned by way of a daiabase, relational database, software program, spreadsheet, file partition, or the like.
  • the data sources may also be connected within or between one another by way of a network., electrical, or optical connection.
  • Clinical dat 803 that may include, tor example, further medical observations of the patient, along with Electrocardiogram data 805, historical data. 807 such as prior syncope data, and genetic testing data 809, are provided .
  • Treatment constructs may be developed or specified by way of a computer or microprocessor based system.
  • FIG. 9 is a diagram depicting a method of determining LQT 1 patient responders and non-responders to beta blocker therapy.
  • a pred.omin.ani medical treatment available for the management of LQTl patients is beta-blocker therapy.
  • Beta blockers are beta-adrenergic blocking agents, beta-adrenergic antagonists, beta-adrenOreceptor antagonists, or beta antagonists that are considered a class of drugs. Examples of beta blockers include, but are not limited to.
  • a method of formulating the composition of a therapeutic agent for treatment of genetic long QT syndrome may include the steps of performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indi.cati.ve of long QT syndrome, identifying with genetic testing the specific types of genetic mutations that are indicative of long QT syndrome, separating. KCNQ1. channel.
  • compositions of a therapeutic agent for treatment of genetic long QT syndrome may include the steps of performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indicative of long QT syndrome, identifying with genetic testing the specific types of genetic mutations that are indicative of long QT syndrome, separating KCNQ 1 channel C-Ioop missense mutations from other mutations in the K.CNQ!
  • a method of quantifying optimal therapeutic efficacy of a drug .for treatment of congenital long QT syndrome may include
  • a method of measuring a response to beta, blocker therapy in patients with long QT syndrome may include the steps of performing genetic testing on patients suspected of having long QT syndrom to identify genetic mutations indicative of long QT syndrome, identifying by way of genetic testing K.CNQ.1 channel C-Joop missense mutations in genetic testing results, and correlating K.CNQ1 channel C-ioop missense mutations with an improved response to beta blocker therapy over other .mutations responsible for genetic long QT syndrome.
  • Figure 10 is a diagram depicting a method of determining LQ 1 patients at increased or lower risk of exercise triggered events.
  • Our findings show that patients with C-Ioop mutations have increased risk for arrhythmic events during exercise, whereas other LQT.1 mutations are indicative of a significantly lower risk for exercise-triggered events. This data may change current recommendations for sports participation in patients with inherited arrhythmic disorders.
  • Figure 0 further describes our method where an LQT.l patient .1001 undergoes genetic testing to generate genetic testing data 1003 related to lon QT syndrome.
  • the presence of C-ioop mutations 1005 are indicative of increased risk of arrhythmic events during exercise; whereas LQTI patients with no C-Ioop mutations show lower risk of exercise triggered events 1009.
  • Figure 1 1 is a diagram depicting a method of determining female LQTI patients at increased risk of sudden cardiac death.
  • Prior studies suggest that LQ ' T! women have a Sower risk of cardiac events than. men.
  • Our new data sho ws that LQTI women with C ⁇ loops mutations have a very high-risk of sudden cardiac death that is similar to that of men. This new finding has important implications for the management and risk assessment of an important subgroup of LQTS lor whom current information, regarding arrhythmic risk is very limited.
  • Figure 1 1 further describes our method where a female LQTI patient 1 101 undergoes genetic testing to generate genetic testing data 1 103 related to long QT syndrome.
  • C-Ioop mutations 1 105 are indicative of a similar risk of cardiac events as compared to male LQTI patient 1 107; whereas female LQTI patients with no C-Ioop mutations show lower risk of cardiac events than comparable male LQTI patients 1 1.09.

Abstract

Systems And Methods for risk stratification, therapies, targeted treatment and prevention of life-threatening ventricular tachyarrhythmias and sudden cardiac death for patients with congenital type-1 long QT syndrome (LQT1) is disclosed. Genetic testing to identify genetic mutations indicative of long QT syndrome is performed, with KCNQl channel C-loop missense mutations being separated from other mutations. The KCNQl channel C-loop missense mutations are an indicator of improved response to beta blocker therapy over other mutations responsible for genetic long QT syndrome. The use of such genetic testing data and targeted risk stratification is useful in determining the composition of a therapeutic agent, taking a therapeutic action, determining the optimal therapeutic efficacy of a drug for the treatment of long QT syndrome, determining a response to beta blocker therapy for patients with genetic long QT syndrome, and the like.

Description

ί
STRATIFICATION, THERAPIES, TARGETED TREATMENT AND
PREVENTION OF LIFE-THREATENING VENTRICULAR TACHYA
This application claims priority to United States Patent Application Serial No. 61/483,626 filed May 6, 201 ί entitled "METHOD AND SYSTEM FOR RISK. STRATIFICATION, THERAPIES, TARGETED TREATMENT AND PREVENTION OF LIFE-THREATENING VENTRICULAR TACHYARRHYTHMIAS A D SUDDEN CARDIAC DEATH FOR PATIENTS WITH CONGENITAL TYPE- 1 LONG Q'F SYNDROME (LQT'O by Bar Sheshet, Golden berg. Lopes, Moss, an d OuchL
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This .invention was made with United States government support under HL-33843 and HL-51618 awarded by National Institutes of Health. The government of the United States has certain rights in the invention.
TECHNICAL FIELD
This invention relates generally to systems and methods for risk stratification, therapies, targeted treatments and prevention of cardiac disorders, and more specifically to Systems And Methods For Risk Stratification, Therapies, Targeted Treatment And Prevention of Life- Threatening Ventricular Tachyarrhythmias And Sudden Cardiac- Death For Patients With Congenital Type- 1 Long QT Syndrome (LQT1 ),
BACKGROUND ART
Congenital long-QT syndromes (LQTS) are inherited arrhythmogenie disorders that increase the risk for ventricular tachyarrh tmias and sudden cardiac death in young individuals without structural heart disease, LQTS type- 3 (LQT!) is the most common genetic form of these disorders. Patients with LQTl have increased risk for life-threatening ventricular tachyarrhythmias and sudden cardiac death during sympathetic activation, associated predominantly with exercise activity or swimming. Accordingly, current guidelines recommend non-specific treatment wit beta-blocker therapy in. LQTS patients. Despite this, however, there are still a considerable number of LQTl patients who experience life-threatening ventricular tachyarrhythmias during beta-blocker therapy. Furthermore, despite multiple attempts for risk stratification studies in long QT syndrome, currently there is limited ability to identify LQTS patients who will not respond to beta-blocker therapy .
The Applicants have recently discovered that mutations in the cytoplasmic interconnecting loops C- loops) of the potassium channel result in severe impairment of sympathetic regulation of heart cells. The Applicants have determined from both expression studies and iranslational clinical studies the following important findings: 1) patients with missense mutations located in the C-loops exhibit the highest risk for life threatening cardiac events (including aborted cardiac arrest and sudden cardiac death), independentl of clinical and electrocardiographic variables; 2) beta-blocker therapy is associated with a pronounced reduction in the risk of life-threatening cardiac events among carriers of missense C-loop mutations, whereas the benefit of this mode of medical therapy is significantly attenuated in LQTl patients with other mutations;. 3) mutations in the C-loops specifically increase the risk of arrhythmias during exercise activity: 4) C-loop mutations can be used to distinguish between LQTl women with a high or low risk of sudden death. These findings, as well as other disclosed herein, have important implications for improved risk stratification, identification of response to therapies, and novel management strategies for the prevention of sudden cardiac death in LQTl patients and possibl also patients of a more general population.
It is thus an object of the present invention to provide a method for risk stratification tor sudden cardiac death in LQTl patients based on mutation function. It is another -object of the present invention to provide improved therapies by identifying responders and non-responders to beta-blocker therapy. It is another object of the present, invention to provide targeted therapies for long-QT syndrome that are mutation specific. It is yet another object of the present invention to provide improved guidance for LQT1 patients with, regards to physical activit and competitive sports. It is another object of the present invention to provide improved risk assessment and risk management for women with long-QT syndrome. It is another object of the present invention to provide identification and management of at risk, individuals in the general population who are at increased risk of arrhythmic events. It is yet another object of the present invention to provide a method for formulating a composition of a therapeutic agent for treatment of genetic long QT syndrome. It .is another object of the present invention to provide a system for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden cardiac death in patients with congenital type- 1 lon QT syndrome (LQ Il ). It is another object of the present invention to provide method of targeted risk stratification and treatment for use in defining a therapeutic action to be taken, ft is another object of the present invention to provide a method of quantifying optimal therapeutic efficacy of a drug for treatment of congenital long QT syndrome, it is another object of the present invention to provide a method of measuring a response to beta, blocker therapy in patients with long. QT syndrome. These and other objects of the present invention are not to be considered comprehensive or exhaustive, but rather, exemplary of objects that may be ascertained after reading this specification and claims with the accompanying drawings.
DISCLOSURE OF THE INVENTION
In accordance with the present invention, there is provided Systems And Methods for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden 5 cardiac death tor patients with congenital type-1 long QT Syndrome (LQTl) comprising an instrument for genetic testing of LQTl syndrome and a treatment construct using data from genetic testing for therapy of LQTl syndrome.
The foregoing paragraph has been pro vided by way of introduction, and is not intended to limit the scope of the invention as described in this specification, claims and the attached ] o drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described by reference to the following drawings, in which .like numerals refer to like elements, and in which:
Figure I is a diagram depicting frequency and location of mutations in the CNQ1 Potassium Channel from a study of 860 subjects;
Figure 2 is a graph of Kaplan-Meier estimates of cumulative probability of a life threatening cardiac event by mutation location and type;
Figure 3 is a bar graph depicting risk of life threatening cardiac events by mutation location, and beta blocker treatment; Figure 4 depicts regulation of LOT.! mutant channels by PKA;
Figure 5 is a diagram depicting risk stratification for A.CA or SCD in LQTS patients;
Figure 6 is a diagram depicting risk, stratification for ACA or SCD in LQl'l patients according t the presen i invention ;
Figure 7 is a diagram depicting methods of targeted treatment of the present invention;
Figure 8 is a diagram depicting data sources used for treatment, constructs of the present invention;
Figure 9 is a diagram depicting a method of determining LQT1 patient responders and non- responders to beta blocker therapy:
Figure 10 is a diagram depicting a method of determining LQTI patients at Increased or lower risk of exercise triggered events; and
Figure 1 .1 is a diagram depicting a .method of determining, female LQT.I patients at increased risk of sudden cardiac death. The present invention will be described in connection with a preferred embodiment, however, it will be understood that there is no intent to limit the invention to the embodiment described. On the contrary, the intent is to cover all alternatives, modifications, and 'equivalents as may be included within the spirit and scope of the invention as defined by this specification, claims and the attached drawings.
BEST MODE FOR CARRYING OUT THE INVENTION
Congenital long QT syndrome is characterized by way of an electrocardiograph (ECG) where there is a prolonged QT interval manifested by ventricular arrhythmias that may result in life -threatening cardiac events and sudden cardiac death. Current treatment for congenital long QT syndrome includes beta-blockers, implantable cardioverter-defibrillators fTCDs), and surgical left cervicothoracie sympathetic denervation (LCSD). While beta-blocker therapy is considered a first line of therapy, and some practitioners admimster beta-blockers to all long QT syndrome patients, there i s a subset of individuals with long QT syndrome genotypes where beta-blocker therapy is not effective and they remain symptomatic. There remains a need for additional long QT syndrome therapies that may require a more targeted approach to treatment.
Genetic diagnostic testing for long QT syndrome has begun to provide identification of congenital long QT syndrome, and may be used to identity individuals with congenital long QT syndrome that are not diagnosed with traditional methods. Through extensive research with LQTl genoryped individuals with varying genetic mutations, a Systems And Methods For Risk Stratification- Therapies, Targeted Treatment And Prevention of Life-Threatening Ventricular Tachyarrhythmias And Sudden Cardiac .Death For Patients With Congenital Type-l Long QT Syndrome (LQTl ) has been developed. Long QT syndrome type-l can be identified by mutations in the KCNQ1 gene encoding. Identification of mutations in the cytoplasmic loops of the KCNQ1 channel have been identified and used for the method and systems described herein.
Long QT syndrome type-l (LQTl) is the most common type of hereditary long QT syndrome, making up about 30 to 35 percent of genotyped patients (Goldenberg L Moss AX Long QT syndrome. Journal of the American Colege oft Cardiology.. 2008;51 :2291-2300). LQTl arises from a decrease in repo!arizing potassium current due to mutations in the KCNOJ gene encoding the a suimo.it of the slowly activating potassium channel ϊκ». Exercise is the main trigger for cardiac arrhy thmic events in patients with LQTl (Schwartz Pi, Priori SG, Spazzolini C. Moss AJ, Vincent, GM, apoiitano C, Denjoy L Guicheney P, Breithardt G, Keating MT, Towbin JA. Beggs AH, Brink P, Wilde AA, Toivonen L, Zareba Ws Robinson JL, Timothy Corfield V, Wattanasirichaigoon D, Corbett C, Haverkamp W, Schutee-Bahr Bt Lehmann MR, Schwartz , Coumel P, Bloise R. Genoiype-phenotype correlation in the long-QT syndrome; Gene-specific triggers for life-threatening arrhythmias. Circulation. 2001 ;1.03:89- 95). Activation of βΐ -adrenergic receptors (βΙ-AR) is the major signaling pathway contributing to increase in heart rate and cardiac output during exercise. βΙ-AR activation leads to activation of protein kinase A (P A). which directly phosphorylates the KCNQi suhunit, increasing 1Ks function (Walsh KB, Kass RS. Regulation of a heart potassium channel b protein kinase A. and C Science. 1988;242:67-69. Also,. Marx SO, KLurokawa J, Reifcen S, Motoike H, D'Armiento J, Marks AR, Kass RS. Requirement of a macromoiecular signaling complex for beta adrenergic receptor modulation of the KCNQI -KCNE l potassium channel. Science. 2002;295:496-499)
The increase in IK* is thought to suppress the premature beats and afterdepolarization induced by Increased Ca** currents during (^-adrenergic stimulation (Shimizu W, Antzdevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQTl , I.QT2 and. LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35:778-786). Accordingly, 8-biockers are the treatment of choice for cardiac events in LQTl patients. Data .from several prior long QT syndrome (LQT.S) studies(Goldenberg I, Bradley J, Moss A. McNitt S, Po!onsky S, Robinson JL„ Andrews M, Zareba W, Beta-blocker efficacy in. high-risk patients with the congenital long-QT syndrome types I and 2: implications for patient management. J Cardiovmc Electrophysioi. 20 0;21 :893-901. Also, Go!denberg f Mos AT Long QT syndrome. ./ Am Coil Cardiol 2008;51 :22 1-2300} demonstrate that despite the significant reduction in the risk of cardiac events and sudden death with B-blocker therapy among LQTl patients, there is still a residual, event rate among high-risk patients who are being treated with this mode of medical therapy, suggesting that 8- b cfcers may be less effective in certain subgroups of LQTl patients.
The primary translated protein (isoform 1) of KCNQI consists of 676 amino acid residue with an intracellular N-terminus region, 6 membrane-spanning segments with two connecting cytoplasmic loops (C-loops) and an intracellular C-terniinus region (Jespersen T, Grunnet M, Olesen SP, The KCNQi potassium channel: From gene to physiological function. Physiology (B ihesdah 2005;20:408-416). Prior genotype-phenotype studies have provided important information regarding the effect of location and coding type of the channel mutations on the phenotypic manifestations and clinical course of LQTl patients. These studies have shown that missense mutations and mutations located at the transmembrane region (including the C-loops) were associated with greater risk for cardiac events (Moss AX Shimizu W, Wilde A A, Towbin 1A, Zareba W, Robinson JL, Qi M, Vincent GM, Ackerman Ml, Kaufman ES, Hofman N, Seth Rf amakura S, Miyamoto Y, Goklenberg I, Andrews ML, McNitt. S. Clinical aspects of iype-1 long-QT syndrome by location, coding type, and biophysical function of mutations involving the kcnq i gene. Circulation. 2007; ί 15:2481-2489). However, the mechanism related to the increased risk associated with transmembrane mutations has not been studied. C-Ioops in the transmembrane region were suggested to affect adrenergic channel regulation by PKA (Matavel A, Medet E, Lopes CM. P A and P C partially rescue long QT type 1 phenorype b restoring channel -PIP2 interactions. Channels (Austin). 2010;4:3-1 1). We therefore hypothesized that LQT1 patients with C-loop mtssense mutations would exhibit a differential clinical risk and response to B-bloeker therapy compared with carriers of other mutations in the KCNQJ channel. Accordingly, we conducted a new study that was carried out in a large cohort of subjects having a spectrum of KCNQ1. mutations from the International LQTS Registry, and was designed to: 1) investigate the clinical outcomes among J CNQl mutation carriers by further dividing the transmembrane region into membrane spanning and C- !oop domains; 2) determine a possible differential response to B-bfocker therapy depending o mutation location and function related to PKA regulation; and 3) relate the clinical data to functional studies of changes in function and β-AR regulation In mammalian cells. Methods
Study Population.
The study comprised 860 patients with genetically confirmed KCNQ1 mutations derived from 170 proband -identified families. The proband in each family had QTc prolongation not due to a known secondary cause. The subjects were drawn from the Rochester (a ~ 637), the Netherlands (n ::: 94), the Japanese (n :::: 82), the Danish (n :::: 43), and the Swedish (n:::4) portions of the Multieenter Mutation Registry. All .subjects or their guardians provided informed consent for the genetic and clinical studies,. Patients with congenital deafness or patients with multiple LQTS associated mutations were excluded from the study. Phenotype characterization
Routine clinical and electrocardiographic parameters were acquired at the time of enrollment in each of the registries. Follow-up was censored at age 41 years to minimize the influence of coronary disease on cardiac events. Measured parameters on the first recorded ECG included
QT and R-R intervals in milliseconds, with QT corrected for heart rate by Bazeti's formula. Clinical data were collected on prospectively designed forms with information on demographic characteristics, personal and family medical history, electrocardiographic findings, therapy, and end points during long-term follow-up. Genotype characterization
The KCNQl mutations were identified with the use of standard genetic tests performed in academic molecular-genetic laboratories. Genetic alterations of the amino acid sequence were characterized by location and by the specific type of mutation (m!ssense, splice site, in-frame insertions/deletions, nonsense, stop eodon, and fratneshiit).
We evaluated the risk associated with 4 main prcspeeified subgroups: 1) C- or N-termmus- missense; 2) membrane spamiing-misscnse 3) C-loops-tnissense; and 4) non-missense (I.e. splice sites, in-frame insertions, in-frame deletions, stop codons, and frameshift). The membrane spanning region of the KCNQl -encoded channel was defined as the coding sequence involving amino add residues between 120-170 (SI-S2), 196-241 (S3-S4), and 263- 355 (S5-S6), with the C-loops region between residues 1 71-195 (S2-S3) and 242-262 (S4-S5), as depicted in Figure I . The N-terminus region was defined before residue 120 and the C- terminus region after residue 355. 'Figure 1 is a diagram depicting -frequency and locaiion of mutations in the KCNQl Potassium Channel from a study of 860 subjects. In Figure I , the a subunit involves the ' -terminus (N), 6 membrane-spanning segments, 2 intraeytoplasmie loops (S2-S3 and S4-S5) and the C-temiinus portion (C). The size of the circles in Figure 1 reflect the number of subjects with mutations at the respective locations., Cellular Expression Studies
In order to study the mechanism underlying the risk for cardiac, events in patients with rnissense C-ioop mutations as compared with other rnissense mutation located in ditlerent domains of the channel, we measured channel function and regulation for channels formed with two mutant subunits present in C-loops (V254M and 243C) and two mutant subunits present in the membrane spanning domain (G.16S and S225L). The mutations chosen included the most common, mutations in each domain in the LQT1 registry, Wild type-and mutant KCNQl subunits, together with KCNEl subunits cDNA. were transacted into HEK293T cells. Mutant KCNQl cDNA was iransfeeted in combination with WT-KCNQ 1 to mimic the heterozygous nature of the disease (WT-KCNQl :mutant KCNQl :KCN B 1-0.5:0.5:1). We measured ion channel currents, in the presence and absence of f orskoim, a protein kinase A activator. Details of the molecular biology and electrophysiological methods are described further later in this specification. End point
The primary end point of the study was {he occurrence of a first life-threatening cardiac event, comprising aborted cardiac arrest (ACA) requiring external defibrillation as part of the 5 resuscitation) or LQTS-related sudden cardiac death (SCD) abrupt in onset without evident cause, if witnessed, or death that, was not explained by any other cause if it occurred in a non- witnessed setting). The consistency of the results among patients who received an implantable cardioverter defibrillator (ICD) during foilow-up was evaluated in a secondary analysis that included the occurrence of a first appropriate ICD shock in the composite ACA or SCD end ] o point.
Statistical analysis
Characteristics of the 4 subgroup of" patients categorized by mutation location and type were compared with the one way ANOVA test or Chi square and Fisher exact tests, as appropriate,
15 The probability of a first life-threatening cardiac event by the mutation-location and type subgroups was graphically displayed according to the method of Kaplan and Meier, with comparison of cumulative probability of events by the log-rank test. The Cox proportional- hazards sun'ivorship model was used to evaiisate the independent contribution of clinical, and genetic factors to the first occurrence of a Hfe-ihreatening cardiac event, from birth through age 0 40 years. The Cox regression models, stratified by decade of birth year and allowing for time- dependent covariat.es, were fitted to estimate the adjusted, hazard ratio function of age (Hobbs JB, Peterson DR, Moss AJ, McNitt S, areba W, Goldenberg I, Qi M, Robinson IL, Sauer AJ, Ackerman MJ, Benhorin T Kaufman ES, Locati EH, Napolitano C, Priori SG, Towbin JA, Vincent. GM, Zhang L. Risk of aborted cardiac arrest or sudden cardiac death during 5 adolescence in the kmg-QT syndrome. Journal of the American Medical Association.
2006;296:1249-1254). Therefore, to fulfill the assumption of proportional, hazards for sex over the entire age range, a time-dependent covariate for sex (via an interaction with time) was incorporated, allowing for different hazard ratios by sex before and after age 1.3 years. Patients who did not have an ECG for QTc measurement (n=T27) were identified in the Cox models as 0 "QTc missing", and ail Cox models were adjusted for this QTc missing parameter. The influence of time- dependent B-blocker therapy (the age at which β- blocker therapy was initiated) on outcome in the subgroups of patients with and without. C-loop-missense mutations was determined by adding "time-dependent β-bloeker ^-by-^mutation category" interaction term to the multivariate Cox model Since almost all subjects were first- and second-degree relatives of probands, we adjusted for the effect of potential lack of independence between subjects using the robust sandwich estimator for family membership. We have carried out the following additional secondary analyses: 1) Including the biophysical function of the mutaiions (categorized as dominant-negative, haploinsuf icieney, and unknown) (Moss A J, Shlmtzu W, Wilde AA, Towbin J A, Zareba W, Robinson II... Qi M. Vincent GM, Ackerman MJ, Kaufman ES, Hofraan N, Seth R, Kamakura S, Miyamoto Y, Goldenberg L Andrews ML, McNitt S. Clinical aspects of type- 1 kmg-QT syndrome by location, coding type, and biophysical function of mutations involving the kenql gene. Circulation. 2007; 115:2481 -2489) as a covariate in the model, 2) excluding the large subgrou of patients with V254M mutations, and 3} including appropriate ICD shocks in the composite end point. The statistical software used for the analyses was SAS version 9.20 (SAS institute ine, Gary. NC). A 2-sided 0.05 significance level was used for hypothesis testing. Additional statistical methods regarding- molecular biology and expression studies are provided herein. Results
Study population
The spectrum of mutations as categorized by location and type and their respective number of carriers are presented in "fable I below, "Hie location, and. frequency of missense mutations is presented diagramniatlcally in Figure 1. Of the total 100 different KCNQ1 mutations identified, 79 were missense mutations and 21. non-missense mutations. Missense mutations were further categorized according to their location: there were 29 different, mutations in C-temiinus or N- terrninas regions (27 in C~terminus), 34 mutations in membrane spanning regions, and 16 mutaiions in the C-l op regions (8 in S4-S5 loop and 8 in S2-S3 loop). Material and Methods Molecular Biology
Human KCNQ1 and KCN.EI in pCD A3.1 (†) vector were gifts from Dr. Robert S. Kass (Columbia University, New York, NY). PCR based site direct, mutagenesis was performed using FPU ultra DNA polymerase (Stratagene, La Jolla CA), Construct sequences were confirmed by DNA sequencing (Cornell University, Ithaca, NY and University of Rochester, Rochester, Y). Eketrophysiotogy
KCNQ1 :KCNE1 DNA was expressed either at a ratio of 1 :1 or 0.5:1 in order to mimic the hapioinsuffieient phenolype in HEK293T cells. Each mutant KCNQ.I was expressed in combination with WT-KCNQJ t mimic heterozygous mutation (WT-KCNQl :mutant KCNQl;K£NEI:~Q.5:Q.5: l}. Each mutant current was compared to the current observed from hapioinsuffieient control channel. (0.5 ng Ψϊ-KCNQI: I ng KCNE1) from the same passage cells. Cells were also co-iransiected with 0.2 ng pEGFP-Nl (Clontech, La Jolia, CA) for positive identification of transfected cells by fluorescence. All the transfecfion was done by using Fugene HD trasnfection kit (Roche, Mannheim, Germany). Cells were re-plated on small glass cover slide (VW1L West Chester, PA) coated with 0.02 % gelatin 24 hours after transfectkm by using Accutase (Innovative Ceil technologies, inc., San Diego, CA) and use For experiment 48 hours after transfection.
Currents were activated by 2s depolarizing pulse to τ20 mV every lOsec from a -80mV holdin potential. These were followed by a step to -2()mV. One-way ANOVA followed by Dur ett's Post Hoc test was applied for the assessment of statistical significance. All experiments were performed at room temperature.
Table Is, Distribution of Mutation Location a
Figure imgf000017_0001
Figure imgf000018_0001
The clinical characteristics of patients in the 4 mutation locatkm type subgroups ate presented in Table 1 below. Of the 860 study subjects, 20 percent had C/N temitnai-missense mutations, 44 percent had membrane spanning-rnissense mutations, 15 percent had C-Soop-missense miUations, and 22 percent had non-missense mutations. Patients with C-loop-missense mutations exhibited the longest QTc interval at enrollment, were treated with beta-blockers more frequently during follow-up, and had a significantly higher frequency of cardiac events of any type, including, syncope, ACA, and LQTS death, as compared with the other mutation, subgroups.
Tank 1. Demographic antl clinical chars icteristies
Non-
Missense
misseiise
C/ -Teritt membrane C-loops P-
Parameter
spanning valute 1
Patients (n, %} 173 (20.1) 376 (43.7) 125 (1 ,5) 186 (21.6) - 1
Female (n, %) I 94 (54,3 ) 221 (58.8) 70 (56.0) 1 19 (64.0) 0.28 j
QTc at enrollment, msec 467i63 480efc51 503±S8 470±41 <o.ooi i
Figure imgf000019_0001
*127 patients did not have an ECCi for QTc measurement.
Clinical outcome of patients according to mutation location a d type
Figure 2 is a graph of Kaplan-Meier estimates of cumulative probability of a life threatening cardiac event b mutation, location and type in 4 subgroups. There was a significantly higher event rate in the C- loop- mi ssense subgroup as compared with the other 3 subgroups (p log rank < 0.001). Thus, at age 40 years the rate of life threatening cardiac events was 33 percent in patients with C-loop-missense mutations as compared with < 16 percent in patients with other mutations, as illustrated in Figure 2, In Figure 2, AC ^ aborted cardiac arrest. LQTS~ long QT syndrome. The numbers in parentheses in Figure 2 reflect the cumulative event rate at that point in time.
The findings from the multivariate analysis for the end point of a first life threatening cardiac event, are shown in Table 2 below. Notably, the adjusted hazard ratio lor C~loop- itilssen.se vs. non-mi ssense mutation was 2.75 (Ϊ Κ009), and there was no statistically significant difference in the risk among, the other mutation location/type subgroups. Table 2. Multivariate analysis: Risk factors for aborted cardiac arrest or sudden cardiac deaiti
Figure imgf000020_0001
Secondary confirmatory analyses showed that patients with C-loop-missense mutations had an adjusted hazard ratio of 2.74 (95 percent confidence interval 1.68 to 4.46 jP<0,001 j) for life threatening events as compared with patients with other mutations, The results were consistent when the biophysical function of the mutations was added as a covariate to the multivariate model or after excluding the large subgroup of patients with V2S4M mutations. The results were also consistent after inclusion of appropriate SCO shocks in the composite end point (adjusted hazard ratios for C-loop-missense mutations vs. non-missense mutations of 2.64 [95 percent confidence interval 1.64 to 4.23; Ρ<0.(Κ)1 ]). β-bloe ktr therapy
Multivariate analysis showed a significant differential effect of β-blocker therap on the outcome of patients with C-loop-missense mutation as compared with those who had other mutations, as summarized in Table 3 below. S-bloeker therapy was associated with a significant 88% reduction (P:;::0.02) in the risk of life threatening events among patients with C-loop- missense mutations, whereas the benefit of β-blocker therapy was significantly attenuated among patients with other .mutations in the KCNQ1 channel (adjusted hazard ratio of 0.82 [P:::;0.68j; P-value for treatinent-by-mutatioii-IocationAype interaction™ 0.04). Figure 3 is a bar graph depicting risk of hie threatening cardiac events by mutation location and beta blocker treatment. Consistent with those findings, the rate of A borted Cardiac Arrest or Sudden Cardiac Death, as depicted in Figure 3, was lowest among patients with C-Ioop-missense mutations who were treated with Beta-blockers and highest among patients with C-l.oop-missen.se mutations who were not treated with beta-blockers (0,17 vs. 1.1 1 per 100 patient -years, respectively), whereas patients with other mutations in the CNQ1 channel exhibited intermediate and similar rates of ¾ -threatening events with- and without beta-blocker therapy (0.36 and 0.38 per 100 patient -years, respectively. Event rates per 100 person-years were calculated by dividing the number of events during the period of β-hiockcr therapy or the absence of β- b!ocker therapy by person-years, and multiplying the results by 100. ACA™ aborted cardiac death. LQT ™ long QT syndrome. Table 3. M ultivariate .analysis: response to β-bloeker therapy
Figure imgf000021_0001
'The models are adjusted for sex X age. corrected QT category (including missing QT), mutation, type and location category, and time-dependent B-biocker treatment * p for interaction for mutation loeation-by-β blocker treatment =0.04 Cellular Expr ssion Studies
!n order to understand the mechanism, underlying the increase in risk associated with C-loop mutations we measured channel basal function and regulation in four mutant channels associated with LQTL two in the .membrane spanning domains (GI 68R and S225L) and two located in C- loops (V254M 'and R243C). The mutations chosen included the most common mutation in each domain in the LQTl registry (See Table Is). Figure 4 depicts regulation of LQTl mutant channels by FKA. Channel current was strongly decreased for all four mutations studied when compared to wild type subunits (Figure 4A and B), in addition, because activation by FKA is thought to be particularly important for Ks function and to underlie arrhythmogenesis in LQTl (Marx SO, Kurokawa J, R.eiken S. Motoike Fl, D'Armiento J, .Marks AR, .ass RS. Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the CNQl - CNEI. potassium channel Science, 2002:295:496-499, Also, Terrenoire C, Clancy CE, Cormier J W. Sampson J, Kass RS. Autonomic control, of cardiac action potentials: Role of potassium channel kinetics in response to sympathetic stimulation. Circ Res. 2005;96:e25-34. Also, Potet F, Scott JD, Mohammad-Panah R, Escande D. Baro L A AP proteins anchor cAMP-dependent protein kinase to KvLQTl/Is channel complex. Am J Physiol Heart Circ Physiol. 2001 :280; 112038-2045). we measured the effect of die PKA activator Forskolin (Figure 4 A, C and D), Bot C-loop mutations showed dramatically impaired, response to Forskoim,. whereas the mutations located in the membrane spanning domain showed a strong activation by Forskolin. as did the wild type K.CNQ1 channel.
In Figure 4? G l 68R and S225L are missense mutations located in the membrane spanning region: R243C and V254M are missense mutations located in the cytoplasmic loops region. A: Typical ion channel current measured before and after 10 mm application of the PKA activator Forskolin (10 μΜ) for left: wild-type (WT) and right: WT and mutant subunits co-expresssed at a 1. : 1. rati . Currents were activated by 2sec depolarizing steps to +20 mV from a ~80mV holding potential. These were followed by a step to -20mV, B; Average current measured for cells expressing WT and mutant subunits measured at +40 mV after 2see depolari ation. C: Typical time course of current regulation measured at +20 rnV after 2sec depolarization for channels formed by either WT or mutant co-expressed with WT subunits, as indicated. Current was normalized to current in the absence of Forskolin application. D: Summary data for experiments done as in C, KCNE1 was co-expressed with the KCNQ1 subunits at a mol r ratio 1 :1. The present analysis among 860 LQT! patients with a wide range of mutations in the KCNQi channel provides several important implications regarding risk assessment and management in this population: (1) patients with missense mutations located in the C -loops exhibit the highest risk for life threatening cardiac events, independently of clinical and electrocardiographic variables; (2) Beta-blocker therapy is associated with a pronounced reduction in the risk of ACA or SCO among carriers of missense mutations in the C-loop, whereas the benefit of this mode of medical therapy is significantly attenoated in LQT! patients with otiier mutations; (3) expression studies of C-ioop mutations suggest that an impaired regulation by PKA in combination with, basal decrease i channel function is the mechanism underlying the increased risk for cardiac events, and may explain the pronounced response to medical therapy with Beta-blockers among patients with C-loop .mutation carriers.
We e recently shown thai patients with mutations located in the transmembrane region of the channel have a significantly higher rate of cardiac events than those with mutations located m the C-ierminus (Moss AJ, Shimizu W» Wilde AA, Towbin jf A, Zareba W, Robinson JLf Qi M. Vincent QM, Ac.ke.rman MJ, Kaufman ES5 Hofrnan N. Setli R, Kamakura S, Miyamoto Y, Goldenberg 1, Andrews ML, Me Mitt B, Clinical aspects of type- Ϊ long-QT syndrome by location, coding type, and biophysical function of mutations involving the kcnql gene. Circulation, 2007;1 15:2481-2489). In addition, mutations in the transmembrane domain were suggested to be associated with, greater prolongation, in the QTc during exercise (Shimizu W, Horie M, Ohno S, Takenaka K, Yamaguchi M, Shimizu M, Washizuka T, Aizawa. Y, Nakaniura K, Ohe Τ» Aiba 1 Miyamoto Y, Yoshimasa Y, Towbin J A, Priori SG, Kam.ak.ura S. Mutation site-specific differences in arrhythmic risk and sensitivity to sympathetic stimulation in the LQT1 form of congenital long QT syndrome: Multiceiit.er study in Japan. J Am Coll Cardiol. 2004:44; 1 .17-125). However, the mechanisms related to those effects have not been studied. Our study extends these observations and shows that mutations in part of the transmembrane domain, the C-loops, are associated with a longer QTc on. the ECG, and with a significant 2.7-fold increased risk of life threatening events as compared with other mutations in the KCNQI channel (including the .membrane-spanning [non-C-loopj mutations).
The S2-S3 and S4-S5 C-loops have been previously suggested to have an important functional role in modifying the function of voltage gated potassium channels (Lsacoff BY. Jan YN, Jan LY, Putative recepto for the cytoplasmic inactivation gate in the shaker K÷ channel. N ture* 1 91:353:86-90). In particular for the S4-S5 loop has been suggested to .mediate a functional interaction with the auxiliary KCNB1 subunits (Franqueza L, Lin M, Shen J, Splawskl L Keating M , Sanguinetti MC, Long QT■ syndrome-associated mutations in the S4- SS linker of KvLQTl potassium channels modify gating and interaction with mink subunits* J Biol Chem. 1999;274:21063-21070). Most recently, LQT1 mutations in C-Ioops, when expressed in the absence of wild-type subunits, were suggested to affect adrenergic channel regulation (Matavel A, edei E, Lopes CM. PKA and PK.C partially rescue long QT type 1 phenotype by restoring chan.nel-PTP2 interactions. Channels (Austin). 2010;4:3-1 1 ). Our results showed thai even when expressed hi the presence of wild-type subunits, C-loop mutations can dramatically affect channel regulation. It i conceivable thai a decrease in channel regulation, as observed for the€4oop mutations, will lead to an increase i the burden of the mutation during adrenergic stimulus. The increase in cardiac risk associated with C-loop mutations is independent of traditional, clinical variables; this can be explained by a blunted P A-medtated activation because QTc is generally measured at. rest. Thus, our results suggest that exercise may exacerbate the QTc prolongation for C-loop domain mutants.
Current guidelines recommend empiric therapy with β-blockers in all L.QTS patients (Zipes DP, Camm A J, Borggre e M, Buxton AE, Chairman B, Pronier M, Gregoratos G, Klein G, Moss AJ, Myerburg RJ, Priori SO, Quinones MA, Roden DM, Silka ML Tracy C, Smith SC, Jr., Jacobs AK, Adams CD, Anlman EM, Anderson JL, Hani SA, Haiperin JL. ishimura. R, Ornato JP, Page RL, Riegei B, Blanc JJ, Budaj A, Dean V, Deckers JW, Despres C, Dieksiein , Lekakis J. McGregor K, Metra M. Morais J. CXsterspey A, Taniargo JL, Zamorano JL. ACC/ AHA/ESC 2006 guidelines for management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: A report of the ameriean college of cardiolog /american heart association task force and the european society of cardiology committee for practice guidelines (writing committee to develop guidelines for management of patients with ventricular arrhythmias and the prevention of sudden cardiac death): Developed in collaboration with the european heart rhythm association and the heart rhythm society. Circulation. 2(K)6;.l 14:e385-484). The present study shows for the "first time, a mutation- specific response to beta-blocker therap in type-! long QT syndrome, demonstrating that beta- blockers were associated with a significant greater reduction in the risk of life-threatening cardiac events among patients with mutations located in the C- loops as compared with all other mutations. It is conceivable that during β-adrenergic stimulation patients with mutations located in the C- loops have an unopposed increase in inward C ^ currents and prolongation of repolarization due to blunted P A- ediated activation of JKS.~ |3- blockers may decrease these unopposed inward Ca~! currents, shorten repolarization and reduce the risk for ventricular arrhythmias (Huffaker R, Lam ST, Weiss JN, ogan B. Intracellular calcium, cycling, early afierdepolarixations, and reentry in simulated long QT syndrome. Heart Rhythm. 2004; 1 :44 Ϊ- 448), whereas patients with other mutations do not exhibit such an effect.
5 Study limitations
The international LQTS Registry records therapies that are prescribed at the discretion of the treating physicians to enrolled subjects, and therefore β-blocker administration, was not randomized. We have therefore carried out multivariate analyses that used beta -blocker therapy as a time-dependent covariate, and have further evaluated the benefit of medical therapy with
] o beta-blockers within higher- and lower- risk subsets of LQTl patients. Our results trom the time-dependent adjusted models suggest that the benefit of medical therapy is attenuated among lower- isk patients with non-C-loop-missense mutations; however, these lower-risk patients should still, be treated with beta-blocker therapy according to guidelines (Zipes DP, Camm AJ, Borggrefe M, Buxton AE. Chaitman B, Fromer M, Gregoraios G, Klein G, Moss AJ. Myerhitrg
15 RJ, Priori SG, Quinones MA, Roden DM, Silka MJ. Tracy C, Smith SC Jr.. Jacobs AK, Adams CD, Amman EM, Anderson JL, Hunt SA, Halperin JL. Nishimura R, Ornaio IP, Page RL, Riegel B. Blanc JJ, Budaj A, Dean V. Deckers J , Despres Cf Dickstein K., Lekakis J, McGregor , Metra M, Morass J, Osterspey A, Tamargo JL, Zamorano JL, ACC/AHA/ESC 2006 guidelines for management of patients with, ventricular arrhythmias and the prevention of 0 sudden cardiac death: A report of the american college of cardiology /american heart association task force and the european society of cardiology committee for practice guidelines (writing committee to develop guidelines for management of patients with ventricular arrhythmias and the prevention of sudden cardiac death): Developed in collaboration with the european heart rhythm association and the heart rhythm society. Circulation, 2()0( ;1 14:e385-484), since the 5 cumulative probability of ACA or SCD from birth through age 40 years among patients with iion-C-loop-niissense mutations was still considerable (between 1 1 and 16 percent).
We used a combination of clinical analysis and. cellular electrophysioSog experiments to investigate the molecular determinants and mechanism underlying the clinical outcomes of a large cohort of subjects having a spectrum of KCNQI mutations categorized by their code type 0 and location. Patients with KCNQI missense mutations located in the C-loop domains had a significantly greater risk for life threatening cardiac events and gained grater benefit when treated with B-blockers as compared with patients having other KCNQI .missense or non- missense mutations independently of clinical risk factors. We suggest that a combination of decrease in basal function and altered adrenergic regulation of the channel underlies the increased cardiac risk in this subgroup of patients. Mutaiioii-specific information should be assessed and incorporated into the risk assessment and the management of LQT1 patients. Our results highlight the importance of understanding the molecular determinants and mechanisms 5 underlying arrhythmogenesi to identify cardiac risk factors for LQ'T! patients, and have implications for risk stratification and treatment in LOT patients.
Genetic testing, as described herein, includes all forms of testing used to test for and detect genetic disorders. Many forms of genetic testing involve examination of the DMA molecule itself, or may involve biochemical tests for enzym.es, chromosom.es, proteins, or other ] o substances. Genetic testing includes techniques to examine genes or markers near the genes.
Genetic testing is often times performed on a sample collected from a patient, such as a sample of blood, hair, skin, or other tissue. One example of such, collection is that of a buccal smear, where a sample of ceils is collected from, the inside surface of a patient's cheek by way of a swab or brush,
15 The ability to identify specific mutations that are not onl indicative of patients that have LQT1 syndrome, but also contain additional, heretofore unknown information related to LQT.l syndrome provides the scientific basis for new and useful, methods of treatment, devices for improved diagnosis and treatmen devices and methods tor improved risk stratification, methods and devices for targeted therapies, and the use and development of therapeutic drugs 0 and drug delivery systems. Various novel uses of the scientific .information produced by the processes, techniques and equipment described herein has profound utility in targeted treatment, therapies, .risk stratification, and prevention of HiVthreatemng ventricular tachyarrhythmias and sudden cardiac death for patients with congenital type-! long QT syndrome (LQT1).
5 For example. Figure 5 is a diagram depicting risk stratification for Aborted Cardiac
Arrest or Sudden Cardiac Death, in. L.QTS patients without use of the present invention and its various embodiments, Various risk factors are considered in the risk stratification diagram of Figure 5. For example, age, gender, prior syncope, frequency of prior syncope, number of syncope events in a specified time period, QTc variables, and the like. With the use of the 0 present invention and its various embodiments described and envisioned herein, risk stratification is genotype-specific. Figure 6 is a diagram depicting risk stratification for Aborted Cardiac Arrest or Sudden. Cardiac Death in LOT! patients according to the present invention. Mutation specific risk factors are considered in the risk stratification diagram of Figure 6. Current risk stratification in LQTS patients relies mostly on non-specific, clinical parameters, including the duration of the corrected QT interval (QTc) and a history of symptoms, whereas genetic/Lunctional. information is usually not incorporated into risk assessment A. prior study in 200? showed that mutations located in the transmembrane region 5 of the potassium channel are associated with increased risk of cardiac events in LQT'l patients.
This study, however, did. not assess specifically the risk of sudde cardiac death and did. not identify functional mechanisms related to mutation-speclfie risk. Our new data show that only missense mutations within the transmembrane region increase the risk of sudden death while other transmembrane mutations are associated with a significantly lower risk. Thus, while the
] o rate of aborted cardiac arrest or sudden cardiac death from birth through age 40 years among LQTi. patients with missense {"-loops mutations is very high - 33%. those who have other mutations in the transmembrane region have a significantly lower rate of only 13%, Therefore, various methods of targeted risk stratification and treatment for use i.n defining a therapeutic action to be taken can be envisioned. The methods may be manual, or they may, in some
15 embodiments of the present invention, be performed on a computer or a computing device or instrument. Examples of steps to be taken may include, for example, performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indicati ve of long QT syndrome and processing the results of the genetic testing on a computer, identifying by way of genetic testing mutations in K.CNQJ channel ("-loops and processing the results of 0 the genetic testing on a computer, evaluating electrocardiogram patient, information on a computer, evaluating patient clinical history on a computer; and defining a therapeutic action to be taken. Therapeutic- actions may include, but are not limited to, use of a beta blocker, use of an implantable cardioverter-defibrillator, alteration of the operation of an implantable cardioverter-defibrillator (such as, for example, by programming), use of surgical left 5 cervieothoracic sympathetic denervation, or use of pharmacological agents, for example, IKs openers, PKC inhibitors, peptides based on the sequence of the c-loop region, c-loop interacting partners that may restore normal channel regulation, drugs thai mimic c-loop peptide action, drugs that bind to c-loop peptides, and drugs that bind to c-loop peptide binding partners.
Therefore, disclosed is a system for identify ing and reducing the risk of life-threatening 0 ventricular tachyarrhythmias and sudden cardiac death in patients with congenital iype-1 long QT Syndrome (LQTI) comprisin an instrument for genetic testing of LQT'l syndrome and for identifying K.C QJ channel mutations; an instrument for identifying C-loop missense mutations in the K.CNQ1 channel mutations; and a computer lor associating a therapeutic action with identified C-Ioop missense mutations in the KCNQ! channel The -computer may contain listings of therapeutic actions to be taken, as will be further disclosed herein. Therapeutic actions may include, but are not limited to, formulating a pharmacological agent containing a beta blocker, formulating or directing mixing or adding a type of beta blocker to a pharmacological agent, formulation of a pharmacological agent that includes, for example, IKs openers, PKC inhibitors, peptides ba.sed on the sequence of the e~!oop region, c-loop interacting partners that may restore normal channel regulation, drags that mimic c-loop peptide action, drugs that bind to c-loop peptides, or drugs that bind to c-loop peptide binding partners, use of an impS.anta.ble cardioverter-dellbrillator, programming or .modification of an implantable cardioverter-defibrillator, the use of surgical left eervicothoracie sympathetic denervation, or the like. Such a system that correlates the heretofore described genetic testing with, a therapeutic action may operate on a computer such as a laptop computer, a tablet computer, a smart phone, an instrument containing a processor, a wearable device such as a vvrisiwaieh, heart rate monitor, blood pressure monitor, or the like. Therapeutic actions may also comprise alerts from the computer that are activated upon reaching a specified bodily indicator such as a specified heart rate, a specified heart rhythm, a specified pet-cent oxygen saturation in blood, a specified blood pressure, a specified chemical marker in blood, or the like. The term specified is defined to be any value, numerical or otherwise, that is entered or otherwise programmed into the computer, either by a human or through another source, such, as another computer, a database, a calculation or calculations, and the like.
Turning now to Figure 7, there i shown a diagram depicting methods of targeted risk ■stratification and treatment using the present invention, instruments such as processes, techniques and equipment heretofore described for genetic testing and subsequent iden ification of mutations in C -loops of the KCNQ1 channel as show In 701 of Figure 7. ECO findings are also evaluated in step 703. Clinical history is also used in step 705, with the useful output being the definitio and subsequent taking of therapeutic actions In step 707 through treatment construct. Treatment constructs and related therapeutic actions may include, but are not limited to, the use of beta-blockers, the use of an implantable cardioverter-defibrillator (KID), the use of surgical left cervieothoracic sympathetic denervation (LCSD). Additional possible novel therapeutic modalities (which are currently not given in LQTS patients) that, may be developed and/or evaluated as a result of the present invention include the use of various pharmacological agents such as an IKs opener (for example, 1.-364373 from. Merck, Retigablne or e ogabine from Valeant Pharmaceuticals and GlaxoSmithKl e), a PKC inhibitor (for example, Roboxistaurin, a calcium dependent P C inhibitor), peptides based on the sequence of the c- ioop region or c-ioop interacting partners (such as KCNQ1(S6)/ .CNQ 1 (Cterm /KCNEi(C- terra) that may restore normal channel regulation, drugs tha mimic e-loop peptide action such as drugs that can. either bind to c-loop peptides or to c-ioop peptide binding partners, or the like.
Figure 8 is a diagram depicting data sources used for treatment constructs of the present invention. The data sources may, in one embodiment of the present invention, be contained in a data storage device such as a hard disk, solid state memory, an optical device, a magnetic device, a semiconductor device, or the like. The data sources contained on physical media may also be organized or patterned by way of a daiabase, relational database, software program, spreadsheet, file partition, or the like. The data sources may also be connected within or between one another by way of a network., electrical, or optical connection. To provide treatment constructs such as a. treatment regimen 801 , various data inputs are used. Clinical dat 803 that may include, tor example, further medical observations of the patient, along with Electrocardiogram data 805, historical data. 807 such as prior syncope data, and genetic testing data 809, are provided . Treatment constructs may be developed or specified by way of a computer or microprocessor based system.
Figure 9 is a diagram depicting a method of determining LQT 1 patient responders and non-responders to beta blocker therapy. A pred.omin.ani medical treatment available for the management of LQTl patients is beta-blocker therapy. Beta blockers are beta-adrenergic blocking agents, beta-adrenergic antagonists, beta-adrenOreceptor antagonists, or beta antagonists that are considered a class of drugs. Examples of beta blockers include, but are not limited to. Propranolol, Sota!oL Timolol, Pindolol, Pejibutoloi, Pxprenolol Nadolol Carteolol, Bucmdolol, AlprenoioL Eucommia. Carvedilol, Labetalol, and the like. Other beta blockers, such as Bi selective agents, may also be used. Examples include, but are not limited to, Acebutoioi. Atenolol, Betaxolol, Bisoproiol, Ceiiprolol, Esmolo.1, Metoproiol Nebivoiov, and the like. However, through our research, with large population studies we show that some LQTl. patients experience life-threatening events despite being treated, with beta-blockers. Our new information shows for the first time a significant difference in response to beta-blockers between LQTl patients who have C-loop mutations (who experience a pronounced 88% reduction in the risk of aborted cardiac arrest and sudden, cardiac death with beta-blocker therapy ) and those who have other LQTl -associated mutations { who do not derive any significant benefit from beta-blockers). These findings have important implications for recommendations for therapy in LQTl patients that take into account mutation-specific response to therapy. Figure 9 further describes our method where an LQT1 patient 901 undergoes genetic testing to generate genetic testing data 903 related to long QT syndrome,. The presence of C-Ioop mutations 905 are indicative of greater response to beta-blocker therap 907; whereas LQTJ patients with no C'-loop mutations show lesser or no response to beta- blocker therapy 909. Therefore, various methods of formulating the composition of a therapeutic agent for treatment of genetic long QT -syndrome are provided by way of this disclosure. For example, a method of formulating the composition of a therapeutic agent for treatment of genetic long QT syndrome may include the steps of performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indi.cati.ve of long QT syndrome, identifying with genetic testing the specific types of genetic mutations that are indicative of long QT syndrome, separating. KCNQ1. channel. C-Ioop missense mutations from other mutations i the CNQ1. channel and formulating therapeutic agent compositions that include Beta Blockers for reduction of life threatening events m patients with K.C Q1 channel C-Ioop missense mu tations. Another example of a method of formulating the composition of a therapeutic agent for treatment of genetic long QT syndrome may include the steps of performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indicative of long QT syndrome, identifying with genetic testing the specific types of genetic mutations that are indicative of long QT syndrome, separating KCNQ 1 channel C-Ioop missense mutations from other mutations in the K.CNQ! channel, and formulating tlierapeutic agent compositions that do not include Beta Blockers for reduction of life threatening events in patients with genetic mutations that do not include KCNQ l channel C-Ioop missense mutations. Other methods are evident after reading this disclosure, and are included in the spirit and broad scope of the present invention as described and depicted herein. For example, a method of quantifying optimal therapeutic efficacy of a drug .for treatment of congenital long QT syndrome may include
performing genetic testing on patients suspected of having iong QT syndrome to identify genetic mutations indicative of long QT syndrome, identifying by way of genetic testing the specific types of genetic mutations indicative of long QT syndrome, separating K.CNQ 1 channel C-Ioop missense mutations from other mutations in the KCNQ1 channel, and assigning a greater probability of therapeutic efficacy of drugs containing beta biockers to patients with CNQ1 channel C-loop missense mutations than to those patients without KCNQ1 channel C'- loop missense mutations. In addition., a method of measuring a response to beta, blocker therapy in patients with long QT syndrome may include the steps of performing genetic testing on patients suspected of having long QT syndrom to identify genetic mutations indicative of long QT syndrome, identifying by way of genetic testing K.CNQ.1 channel C-Joop missense mutations in genetic testing results, and correlating K.CNQ1 channel C-ioop missense mutations with an improved response to beta blocker therapy over other .mutations responsible for genetic long QT syndrome.
Figure 10 is a diagram depicting a method of determining LQ 1 patients at increased or lower risk of exercise triggered events. Currently, there is a nonspecific recommendation, for all LQ f I patients t avoid participation in competitive sports and intense physical activity. Our findings show that patients with C-Ioop mutations have increased risk for arrhythmic events during exercise, whereas other LQT.1 mutations are indicative of a significantly lower risk for exercise-triggered events. This data may change current recommendations for sports participation in patients with inherited arrhythmic disorders. Figure 0 further describes our method where an LQT.l patient .1001 undergoes genetic testing to generate genetic testing data 1003 related to lon QT syndrome. The presence of C-ioop mutations 1005 are indicative of increased risk of arrhythmic events during exercise; whereas LQTI patients with no C-Ioop mutations show lower risk of exercise triggered events 1009.
Figure 1 1 is a diagram depicting a method of determining female LQTI patients at increased risk of sudden cardiac death.. Prior studies suggest that LQ'T! women have a Sower risk of cardiac events than. men. Our new data sho ws that LQTI women with C~loops mutations have a very high-risk of sudden cardiac death that is similar to that of men. This new finding has important implications for the management and risk assessment of an important subgroup of LQTS lor whom current information, regarding arrhythmic risk is very limited. Figure 1 1 further describes our method where a female LQTI patient 1 101 undergoes genetic testing to generate genetic testing data 1 103 related to long QT syndrome. The presence of C-Ioop mutations 1 105 are indicative of a similar risk of cardiac events as compared to male LQTI patient 1 107; whereas female LQTI patients with no C-Ioop mutations show lower risk of cardiac events than comparable male LQTI patients 1 1.09.
Our findings show a very high risk of arrhythmic events in patients wit mutations in the C-loop region of the potassium channel. Therefore, it is possible thai single nucleotide polymorphisms (that are .more common in the genera! population) are responsible for unexplained syncopal and/or arrhythmic events that are currently left, undiagnosed and untreated. Thus, ©tar methods, systems, and devices can be used in related ways, lor example, as a -possible new test to identify risk of arrhythmic events in non-LQTS patients, and further to develop therapies that can be used for the reduction in the risk of sudden death in the general population.
It is, therefore, apparent that there has been provided, in accordance with the various objects of the present invention. Systems And. Methods For Risk Stratification, Therapies, Targeted Treatment And Prevention of Life-Threatening Ventricular Tachyarrhythmias And Sudden Cardiac Death For Patients With Congenital Type-! Long QT Syndrome (LQTl).
. While the various objects of this invention have been described in conjunction with preferred embodiments thereof, it. is evident that many alternatives, modifications, and variations -will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fail within, the spirit and broad scope of this •specification, claims and the attached drawings.

Claims

What is claimed is:
1. A system for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden cardiac death in patients with congenital type- 1 long QT Syndrome (I.QT1 } comprising:
an instrument for genetic testing of LQ'll syndrome and for identifying CNQ1 channel mutations;
an instrument for identifying C-loop rnissense mutations in the KCNQ1 channel mutations: and
a computer for associating a therapeutic action with identified C-loop rnissense mutations in the KC QI channel.
2, The system for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden cardiac death in patient with congenital type-1 long QT Syndrome (LQT1) as recited in claim 1, wherein the therapeutic action is the formulation of a ph.ar.maco logical agent containing a beta blocker.
3. The system for identifying and reducin the risk of life-threatening ventricular tachyarrlvy mias and sudden cardiac death in patients with congenital type-1 long QT Syndrome (LQT1) as recited in claim. 1 , wherein, the therapeutic action is the formulation of a pharmacological agent selected from the group consisting of I s openers, PKC inhibitors, peptides based on the sequence of the e-loop region, c-loop interacting partners that ma restore normal channel regulation, drugs that mimic c-loop peptide action, drugs that bind to e-loo peptides, and drugs that bind to c-loop peptide binding partners.
4, The system, for identifying and. reducing the risk of life-threatening ventricular tachyarrhythmias and sodden cardiac death in patients with congenital type-1 long QT Syndrome (LQ'il ) as recited in claim 1, wherein the therapeutic action is the use of an implantable cardioverter-defibrillator.
5« The system for identifying and reducing the risk of life- threatening ventricular tachyarrhythmias and sudden cardiac death in patients with congenital type-1 long QT Syndrome (LQTl) as recited in claim lf wherein the therapeutic action is the •programming of an implantable cardioverter lellbri Slater,
6. The system for identifying and reducing the risk of hfe-ihreatening ventricular tachyarrhythmias and sudden cardiac death in patients with congenital type- 1 long QT •Syndrome (LQTl) as recited in claim 1, wherei the therapeutic action is the modification of an imp lantable cardioverter-defibrillator.
7. The system, for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden cardiac death in patients with congenita! type-1 long QT Syndrome (LQTl ) as recited in claim L wherein the therapeutic action is the use of surgical left cervicothoraeic sympathetic denervation,
8. The system for identifying and reducing the risk of life-threatening ventricular tachyarrhythmias and sudden cardiac death in. patients with congenital type-1 long QT Syndrome (LQTl) as -recited in claim 1, -wherein the therapeutic action is the activation of an alert by the computer upon reaching a specified metabolic function.
9. A method of formulating the composition of a therapeutic agent for treatment of genetic long QT syndrome comprising the steps of:
performing genetic testing on patients suspected of having long QT syndrome to identity genetic mutations indicative of lon QT syndrome;
identifying with genetic testing the specific types of genetic mutations that are indicative of long QT sy ndrome;
separating KCNQl channel C-loop missense mutations from other mutations in the
KCNQl. channel; and
formulating therapeutic agent compositions that include Beta Blockers for reduction of life threatening events in patients with KCNQl channel C-loop missense mutations.
10. A method of formulating, the composition of a therapeutic agent for treatment of genetic long QT syndrome comprising the steps of: performing genetic testing on patients suspected of having long QT syndrome to identity genetic mutations indicative of Jong QT syndrome;
identifying with genetic testing the specific types of genetic mutations thai are indicative of long QT syndrome;
separating KCNQI channel C-loop missense mutations from other mutations in the KCNQI channel; and
formulating therapeutic agent compositions that do not include Beta Blockers for reduction of life threatening events in patients with genetic mutations that do not include KC Q.J channel C-ioop missense mutations.
1 L A method of targeted risk stratification, and treatment for use in defining a therapeutic action to be taken comprising the steps of:
performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indicative of long QT syndrome and processing the results of the geneiic testing on a computer;
identifying by way of genetic testing mutations in KCNQI channel C-loops and processing the results of the genetic testing o a computer
evaluating electrocardiogram patient information on a computer;
evaluating patient clinical history on a computer; and
defining a therapeutic action to be taken.
12. The method of claim 1 1 , wherein the therapeutic action to be taken is defined to be the use of a beta blocker.
13. The method of claim 1 1 , wherein the therapeutic action to be taken is defined to be the use of an implantable cardioverte -defibrillator.
14. The method of claim. 11, wherein the therapeutic action, to be taken is defined to be alteration of the operation of an implantable cardioverter-defibrillator
15. Th method of claim 1 1 , wherein the therapeutic action to be taken is defined to be the use of surgical left cervicoihoracic sympathetic denervation.
1 .. The method of claim ! 1 , wherein the therapeutic action to be taken is defined to be the use of a pharmacological agent selected from the group consisting of IKs openers, P C inhibitors, peptides based on the sequence of the c-loop region, c-ioop interacting partners that may restore normal channel regulation, drugs that mimic c-loop peptide action, drugs that bind to c-loop peptides, and drugs that bind to c-loop peptide binding partners.
17. A method of quantifying optimal therapeutic efficacy of a drug for treatment of congenital long QT syndrome, the method comprising the steps of:
performing genetic testing on patients suspected of having long QT syndrome to identify genetic mutations indicative of long QT syndrome;
identifying by way of genetic testing the specific types of genetic mutations indicative of long QT sy ndrome;
separating CNQ1 channel C-loop missense mutations from other mutations in the KCNQ1. channel; and
assigning a greater probability of therapeutic efficacy of drugs containing bet blockers to patients with KCNQ'l channel C-loop missense mirtations than to those patients without CNQ1 channel C-ioop missense mutations,
1 8. A method of measuring a response to beta blocker therapy in patients with long QT syndrome comprising the steps of:
performing genetic testing on patients suspected of having long QT syndrome to identif genetic mutations indicative of long QT syndrome;
identifying by way of genetic testing KCNQl channel C-loop missense mutations in genetic testing results; and
correlating KCNQ'l channel C-loop missense mutations with an improved response to beta blocker therapy ove other mutations responsible for genetic lon QT syndrome.
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