CYP2j2*7 genetic polymorphism associated with proarrhythmia and drug-induced proarrhythmia

Abstract

Disclosed is a method of identifying a subject who has or is predisposed to cardiac arrhythmia, the method comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein the presence of the allele indicates that the subject has or is predisposed to cardiac arrhythmia.

Description

CYP2J2*7 GENETIC POLYMORPHISM ASSOCIATED WITH PROARRHYTHMIAAND DRUG-INDUCED PROARRHYTHMIAFIELD OF THE INVENTION

[0001] The present invention generally relates to a method of diagnosing cardiac arrhythmia based on a genetic polymorphism. The present invention also relates to a method of diagnosing cardiac arrhythmia by genotyping CYP2J2*7.BACKGROUND

[0002] Cytochrome P4502J2 (CYP2J2), a member of the cytochrome P450 superfamily, is a membrane-bound monooxygenase primarily localized in the endoplasmic reticulum that utilizes a heme prosthetic group to facilitate biotransformation [1-4], Unlike most cytochrome P450 enzymes (P450s) which demonstrate predominant hepatic expression, CYP2J2 is chiefly expressed in cardiac tissue [5-10] where it emerges as the most abundantly expressed P450 [5,6], Notably, it also serves as the dominant cardiac epoxygenase, with mRNA expression levels exceeding those of CYP2C8 and CYP2C9, the other core P450 epoxygenases, by over 104-fold [6,8],

[0003] While capable of xenobiotic metabolism characteristic of conventional P450s, CYP2J2 is principally responsible for the epoxygenation of arachidonic acid (AA), the predominant polyunsaturated fatty acid (PUFA) in cardiac membranes [11-13], to four epoxyeicosatrienoic acid (EET) regioisomers (Figure 1), with a minor role in biotransformation of other PUFAs (Table 1). EETs are critical signaling lipids that exert profound cardioprotective effects, including vasodilation via regulation of Na+[14-16], L-type Ca2+[15-17] and KATP [16,18,19] ion channels, anti-inflammation via peroxisome proliferator-activated receptor (PPAR) [20-23] and NF-KB pathways [20,21,23,24], stimulation of angiogenesis [25,26] and mitigation of infarct size upon ischemic-reperfusion injury [27-29], Artificial augmentation of EETs, via CYP2J2 overexpression or curtailing of soluble epoxide hydrolase (sEH)-mediated EET degradation through pharmacological inhibition or genetic ablation, have also been demonstrated to evoke in vivo cardioprotective effects in animal disease models. This encompasses reducing arrhythmic susceptibility upon induction of cardiac stress [23,30] and improving outcomes after adverse cardiovascular events, including ischemia-reperfusion injuries [28,29,31-35], heart failure [36,37], hypertrophy [38,39] and arrhythmia [37,40],Collectively, these findings accentuate the protective role of EETs and CYP2J2 in cardiovascular homeostasis.

[0004] Table 1: Summary of endogenous and exogenous substrates of CYP2J2 and respective metabolic pathways mediated by CYP2J2.

[0005] Specifically, EETs are indispensable in cardio-electrophysiological regulation. Aforementioned, Li et al.

[0023] and Westphal et al.

[0030] showed that CYP2J2 overexpression mitigated arrhythmic risk in transgenic mice models subjected to arrhythmia-predisposing cardiac stress. Ke et al. also showcased that ventricular cardiomyocytes isolated from CYP2J2- overexpressing mice exhibited elevated potassium channel activity, leading to shorter cardiac action potentials

[0056] , Correspondingly, inhibition of CYP2J2-mediated EET synthesis has been increasingly associated with drug-induced proarrhythmia. It has been previously demonstrated that dronedarone, an anti-arrhythmic paradoxically correlated with Torsades de Pointes (TdP) and ventricular arrhythmia particularly in hemodynamically compromised patients

[0057] , caused mechanism-based inactivation (MBI) of CYP2J2-mediated EET biosynthesis and increased arrhythmic risk in both in vitro human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and in vivo persistent atrial fibrillation (AF) canine models [46,58], Upon site-specific deuteration to diminish formation of the reactive quinone-oximeintermediate responsible for MBI and thereby preserving EET levels in situ, dronedarone’s proarrhythmic tendencies were alleviated while preserving its pharmacological effects, indicating that perturbation of CYP2J2-mediated EET synthesis was the underlying mechanistic cause of dronedarone-induced proarrhythmia

[0058] , Building upon this, it was subsequently found that 11 of 16 drugs affiliated with intermediate / high risk of TdP, as per adopted by the Comprehensive in-vitro Proarrhythmia Assay (CiPA)

[0059] , reversibly inhibited CYP2J2-mediated EET synthesis

[0060] , further underscoring the climacteric link between perturbation of CYP2J2-mediated EET synthesis and drug-induced proarrhythmia.

[0006] Like other P450s, CYP2J2 exhibits substantial genetic polymorphism with CYP2J2*7 (rs890293) emerging as the most prevalent variant, exhibiting an allelic frequency fluctuating between 1.1-17% depending on ethnicity [4,9,41,61]. CYP2J2*7 is a single nucleotide polymorphism (SNP) involving a G>T substitution in the proximal promoter 50 base pairs (bp) before the transcriptional start site [4,61]. This mutation interferes with Spl transcription factor binding, resulting in a 48% reduction in promoter activity and a corresponding 30% decrease in plasma concentration of 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), a stable byproduct of 14,15-EET breakdown

[0062] , Subsequently, Yamazaki et al. illustrated that the SNP also reduced translational expression of CYP2J2 in human liver microsomes by 60%

[0052] , Given its pervasiveness and speculated implications on cardiovascular health, numerous clinical studies have probed the association between CYP2J2*7 and various adverse cardiovascular events, although its relationship with arrhythmia remains unexplored (Table 2).

[0007] Table 2: Summary of clinical genetic association studies into the association of CYP2J2*7 with adverse cardiovascular events

[0008] However, despite such fervent interest in CYP2J2*7 and the established proarrhythmic implications of CYP2J2 inhibition, the link between CYP2J2*7 and drug- induced proarrhythmia remains poorly understood. Furthermore, the direct cellular' impact of the SNP on CYP2J2 transcriptional expression, enzymatic activity, and most critically cardioelectrophysiology, has yet to be characterized. Given the theoretically negative impact the SNP poses on CYP2J2 expression and consequently EET synthesis stemming from its interference with transcription, it is hypothesized that CYP2J2*7 carriers may be innately predisposed to an elevated risk of cardiac arrhythmia, and by extension drug-induced proarrhythmia upon exposure to CYP2J2-inhibiting xenobiotics. To validate this hypothesis, in this disclosure, spontaneously contracting wild-type (WT), CYP2J2*l / *7 heterozygous and CYP2J2*7 / *7- homozygous (HM) hESC-CMs were utilized as novel genotype models to characterize the SNP’s effects on CYP2J2 rnRNA expression, enzymatic activity and cardio-electrophysiology. By enhancing comprehension of CYP2J2*7 and its ramifications on cardio-electrophysiology, this study seeks to facilitate more informed clinical decision-making, especially concerning carriers of CYP2J2*7.

[0009] The present disclosure describes a method of diagnosing patients who have or are potentially susceptible to cardiac arrhythmia (intrinsic arrhythmia or drug-induced proarrhythmia) by genotyping CYP2J2*7.

[0010] Tn one aspect, the present disclosure refers to a method of identifying a subject who has or is predisposed to cardiac arrhythmia, the method comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein the presence of the allele indicates that the subject has or is predisposed to cardiac arrhythmia.

[0011] In another aspect, the present disclosure refers to a method of treating or preventing cardiac arrhythmia in a subject having or predisposed to cardiac arrhythmia, comprising: a. obtaining a sample from the subject; b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein when the allele is present, the method further comprising administering a reduced dose or optimizing the dosing regimen of a CYP2J2 reversible inhibitor or irreversible inactivator compared to when the allele i absent, or admini tering a therapeutically efficacious dose of a therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator to the subject.

[0012] In another aspect, the present disclosure refers to a method for establishing a prognosis for a patient having or predisposed to cardiac arrhythmia and treated by a CYP2J2 reversible inhibitor or irreversible inactivator, comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein the presence of the allele indicates poor prognosis.

[0013] In another aspect, the present disclosure refers to a detection reagent for carrying out the method as disclosed herein, wherein said detection reagent is an allele- specific probe or an allele-specific primer.

[0014] In another aspect, the present disclosure refers to a test kit comprising one or more containers containing the detection reagent as disclosed herein and one or more componentsselected from the group consisting of an enzyme, polymerase enzyme, ligase enzyme, buffer, amplification primer pair, dNTPs, ddNTPs, positive control nucleic acid, negative control, nucleic acid extraction reagent, and instructions for using said test kit which instruct that the presence of the CYP2J2*7 allele.

[0015] Advantageously, CYP2.T*7 is associated with an underlying cardiac proarrhythmic potential and with drug-induced cardiac proarrhythmic risk. Genotyping CYP2J2*7 is thus applicable to the (1) diagnosis of patients who have a proarrhythmic substrate and potentially susceptible to drug-induced arrhythmic risk (precision diagnosis), (2) optimization of pharmacotherapy of regulatory approved drugs that arc CYP2J2 reversible inhibitors or irreversible inactivators where CYP2J2*7 carriers are contraindicated (precision medicine) and (3) clinical studies that implicate CYP2J2*7 as an efficacy or safety biomarkers.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0017] Figure 1 shows the summary of major metabolic pathways mediated by CYP2J2.

[0018] Figure 2 shows that the employed differentiation protocol was highly effective and consistent for both WT and HM hESCs. (A) Scheme for development of hESC-CM genotype models, with initial CRISPR-mediated genetic manipulation of H7 hESCs and subsequent directed differentiation of hESCs into cardiomyocytes. (B) Schematic illustration of differentiation protocol, adapted from the GiWi and modified Gi(I / M)Wi methodologies [58,74-76], (C) Representative flow cytometry plots illustrating extent of cardiac marker SIRPa expression in WT and HM hESC-CMs between D25-45. (D) Summary of differentiation efficiency per flow cytometry. (E) Expression levels of cardiac marker TNNT2 were evaluated in WT and HM hESC-CMs between D25-30 of differentiation via RT-qPCR with GAPDH as endogenous control.

[0019] Figure 3 is a bar graph showing the mRNA expression of P450 epoxygenases CYP2J2, CYP2C8 and CYP2C9 in WT and HM hESC-CM cell lines.

[0020] Figure 4 shows that the fluorescent properties of CYP2J2-specific metabolite ER- OXPI were exploited for imaging of CYP2J2 in live hESC-CMs. (A) Schematic representation of enzyme-catalyzed demethylation and subsequent spontaneous hydrolysis of ER-BnXPI to form fluorescent metabolite ER-OXPT. (B) Cytotoxicity assessment of ER-BnXPT in hESC-CMs. (C) Fluorescent imaging of CYP2J2 in live hESC-CMs with ER-BnXPI. Images from the top row were unstained hESC-CMs, while images from bottom row were hESC-CMs after incubation with 0.9 M ER-BnXPI for 30min.

[0021] Figure 5 shows that ER-BnXPI was also utilized for quantitative characterization of CYP2J2 functional activity in live hESC-CMs. Data from 3 biological experimental replicates performed on both WT and HM hESC-CMs was combined and fitted into aMichaelis-Menten model as seen above. Apparent Michaelis-Menten parameters of (Kmand Vmax) of CYP2J2- mediated ER-BnXPI demethylation in hESC-CMs for each biological replicate were derived via flow cytometric analysis of the fluorescent emissions of individual hESC-CMs after incubation with varying concentrations of ER-BnXPI (0 05-25 pM)

[0022] Figure 6 shows the summary of beat-to-beat (BTB) intervals in hESC-CMs. (A) Basal mean BTB interval and BTB interval variability (standard deviation / mean) in respective WT and HM wells prior to treatment with DMSO (negative control) or danazol. (B) Cytotoxicity assessment indicated that danazol was non-cytotoxic in hESC-CMs up to a concentration of 20 LIM. (C) Pre-Treatment and Post-Treatment (24h incubation with danazol) mean BTB interval and BTB interval variability as monitored in respective WT and HM cells.

[0023] Figure 7 is a graphical abstract summarizing the overarching methodologies and key findings of this study.

[0024] Figure 8 shows the genotyping of WT and HM hESCs. (A) Genotyping demonstrating wild-type CYP2J2 homozygosity in WT hESCs (B) Genotyping confirming successful CRISPR-mediated mutagenesis and CYP2J2*7 homozygosity in HM hESCs.

[0025] Figure 9 shows the comprehensive Synthetic Route for ER-BnXPI.

[0026] Figure 10 shows the ’H NMR Spectrum of ER-BnXPI (CDCh). ^NMR (400 MHz, CDCh, 298K) 5 8.63 (d, J = 14.8 Hz, 1H), 8 19 (t, J = 5.5 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.54 - 7.44 (m, 3H), 7.39 (dd, J = 8.4, 6.0 Hz, 4H), 7.27 - 7. 19 (m, 4H), 6.99 - 6.92 (m, 3H), 6.89 (d, J = 2 3 Hz, 1H), 6.48 (d, J = 14.8 Hz, 1H), 5.12 (s, 2H), 4 40 (t, J = 7.6 Hz, 2H), 3.83 (s, 3H), 3.35 (q, J = 5.3 Hz, 2H), 3.10 (t, J = 5.2 Hz, 2H), 2.74 (q, J = 5.5 Hz, 4H), 2.36 (s, 5H), 1.93 (q, J = 7.1, 6.4 Hz, 4H), 1.78 (s, 8H), 1.60 (dp, J = 14.8, 6.9 Hz, 2H).

[0027] Figure 11 shows (A) HPLC Chromatogram and (B) Report for ER-BnXPI.

[0028] Figure 12 shows Mass Spectrometry Spectrum of ER-BnXPI under positive electrospray ionization.

[0029] Figure 13 shows the results of Astemizole Cell Viability assays. hESC-CMs between D11-D15 of differentiation were reseeded in Matri gel -coated 96-well plates at acellular density of 4.0xl04cells / well. Upon maturation (D25-30), hESC-CMs were incubated with varying concentrations of astemizole (0.01-50 pM) for 24 h in a 37nC, 5% CO2 incubator. Cytotoxicity of astemizole in both (A) WT and (B) HM hESCs was then assessed by CellTiter- Glo kit (Promega, US) following manufacturer protocols, with resultant luminescence evaluated with an Infinite 200 microplate reader (Tecan). ICso values, indicating the maximum non-cytotoxic concentration, were derived from the nonlinear regression curve (log(inhibitor) vs. response, 4 parameters) generated by GraphPad Prism 8.0.2 (Dotmatics).

[0030] Figure 14 shows the results of Danazol Reversible Inhibition Assays. Prior to characterization, hESC-CMs were dissociated with Accutase and reseeded onto Matrigel- coated 24-well plates between D9-11 of differentiation, and reversible inhibition assays utilizing danazol as reversible CYP2J2 inhibitor and astemizole as probe substrate were conducted on hESC-CMs between D25-30 of differentiation. hESC-CMs were incubated with 0.5 pM astemizole and various concentrations of danazol (0 - 10 pM) for 24 h in a 37 °C, 5% CO2 incubator. 500 pL aliquots of culture medium were then quenched with 1 mL ice-cold ACN containing 20 nM of buspirone internal standard. The remaining medium was aspirated, and hESC-CMs were dissociated with 0.25% Trypsin-EDTA (ThermoFisher), neutralized with PBS (Cytiva) containing 10% FBS (Cytiva) and centrifuged for 5 min at 200g, 4 °C. ImL of the same quenching solution was utilized to resuspend the cell pellet, prior to cell lysis through shearing with a 27G needle followed by ultrasOoni cation on ice for Ih. Quenched aliquots of lysate and media were centrifuged for 30 min at 18,000g, 4 °C, and 500 pL of supernatant was retrieved, dried and reconstituted in cold ACN. BCA Protein Assay (ThermoFisher) was employed for protein quantification of residual lysate aliquots. Liquid chromatography-tandem mass spectrometry was employed for analysis and metabolite quantification of reconstituted samples (LC / MS / MS, Table 3) The peak-area ratio of O-desmethylastemizole against internal standard buspirone was first normalized against protein concentration, prior to subsequent normalization against the negative control (devoid of danazol) to elucidate the extent of inhibition. (A) O-desmethylastemizole formation (medium); (B) O-desmethylastemizole formation (lysate). As depicted, danazol was validated as a potent and concentration-dependent inhibitor of CYP2J2-mediated astemizole O-demethylation between 1 - 10 pM.

[0031] Table 3: Multiple Reaction Monitoring (MRM) Transitions and relevant mass spectrometer configurations for astemizole, O-desmethylastemizole and buspirone. The LC / MS / MS apparatus utilized was an Agilent 1290 Infinity Ultra-High Performance Liquid Chromatography (UPHLC) (Agilent Technologies, US) system coupled with a QTRAP-5500tandem mass spectrometer (AB SCIEX, Canada). A reverse-phase ACQUITYUPLC BEH Cl 8 column (1.7pm, 2.1mm x 50mm) was utilized to attain chromatographic separation at an operating temperature of 45°C. Analyst 1.7.1 (AB SCIEX) and MultiQuant 3.0.3 (AB SCIEX) were employed to operate the LC / MS / MS system and aid in peak integration and analysis respectively. The mobile phases were composed of 0.1% formic acid in H2O (A) and 0.1 % formic acid in ACN (B), with a flow rate of 0.6 ml / min and injection volume of 3 pL. Elution parameters involved initial gradient elution from 20% to 80% B (0 - Imin), followed by isocratic elution at 100% B (1.01 - 1.5min) and lastly 20% B (1.51 - 2min). Compound ionization was achieved by positive electrospray ionization.

[0032] Figure 15 is a bar graph showing mRNA expression of P450 epoxy genases CYP2J2, CYP2C8 and CYP2C9 in CYP2J2*1 / *1 WT, CYP2J2*l / *7 heterozygous and CYP2J2*7 / *7 homozygous hESC-CMs between D25-30 of differentiation via RT-qPCR with GAPDH as endogenous control. Data are shown as mean ± S.E.M. for three independent experiments. Statistical analysis was performed using 2-tailed Student’s r-test. *P < 0.05.

[0033] Figure 16 is a line graph showing that ER-BnXPI was utilized for quantitative characterization of CYP2J2 functional activity in live hESC-CMs. Data from 3 biological experimental replicates performed on CYP2J2*1 / *1 WT, CYP2J2*l / *7 heterozygous and CYP2J2*7 / *7 homozygous hESC-CMs was combined and fitted into a Michaelis-Menten model. Apparent Michaelis-Menten parameters of (Kmand VmoY) of CYP2J2 -mediated ER- BnXPI demethylation in hESC-CMs for each biological replicate were derived via flow cytometric analysis of the fluorescent emissions of individual hESC-CMs after incubation with varying concentrations of ER-BnXPI (0.05-25 pM).

[0034] Figure 17 shows a summary of bcat-to-bcat (BTB) intervals in hESC-CMs. (A) Basal mean BTB interval and (B) BTB interval variability (standard deviation / mean) in respective CYP2J2*1 / *1 WT, CYP2J2*l / *7 heterozygous and CYP2J2*7 / *7 homozygouscells prior to treatment with DMSO (negative control) or danazol. (C) Pre-Treatment and posttreatment (24 h incubation with danazol) mean BTB interval variability as monitored in respective in respective CYP2J2*1 / *1, CYP2J2*l / *7 and CYP2J2*7 / *7 cells. Statistical analysis was performed using Kruskal- Wallis test, ns, non-significant, *P < .05, **P < .01, ***P < .001.

[0035] Figure 18 is a line graph showing the kinetic characterization of 14,15-EET formation as a readout of CYP2J2 enzymatic activity in hESC-CMs. 14,15-EET formation was quantified by LC-MS / MS following 24-hour incubation of hESC-CMs with increasing concentrations of AA. The rate of 14,15-EET production, normalized to total protein content, was plotted against AA concentration. Data from CYP2J2*1 / *1, CYP2J2*l / *7 and CYP2J2*7 / *7 hESC-CMs was combined and fitted into a Michaclis-Mcntcn model to derive apparent Michaelis-Menten parameters of (Kmand VmaxA) of CYP2J2-mediated AA epoxygenation.

[0036] Figure 19 is a line graph showing calibration of 14,15-EET for its quantitation in cell culture medium.

[0037] Figure 20 shows that CYP2J2*7 is associated with the enrichment of non-AF ECG abnormalities in HF patients, (a) Enrichment of abnormal ECG findings in HF patients compared to demographically matched healthy controls, (b) Presence of the CYP2J2*7 polymorphism is associated with the enrichment of non-AF ECG abnormalities in HF patients, (c) With worsening HF, AF is progressively enriched in CYP2J2*1 / *1 patients, whereas non- AF abnormalities are more prevalent in CYP2J2*l / *7 patients. All p-values were calculated using Fisher’s exact test.DETAILED DESCRIPTION

[0038] Cytochrome P450 2J2 (CYP2J2), as the predominant cardiac epoxygenase, chiefly mediates the biotransformation of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs), which are vital for cardio-electrophysiological regulation. The list of single nucleotide polymorphisms (SNPs) associated with CYP2J2, their allele frequencies, enzyme activities and associated disease states is presented in Table 4 below.

[0039] Table 4. List of single nucleotide polymorphisms (SNPs) associated withCYP2J2, their allele frequencies, enzyme activities and associated disease states

[0040] Amongst its genetic polymorphs, CYP2J2*7, involving a -50G>T single-nucleotide polymorphism (SNP) in the proximal promoter region leading to stunted transcription factor binding, is the most prevalent, and has been associated with various adverse cardiovascular events. As shown, CYP2J2*7 has high allele frequency ranging from 2.1 to 17% depending on the population. The other SNPs of CYP2.12 have low allele frequencies of lower than 1 %. Notably, based on the SG10K data, the allele frequency of CYP2I2*7 is 0.054 (5.4%). CYP2J2*7 has been reported to be associated with asthma, cerebral ischemia, coronary artery diseases (e.g. acute coronary syndrome and myocardial infarction), hypertension and ischemic stroke. However, the direct cellular impact of CYP2J2*7, particularly on cardiomyocytc electrophysiology, remains unexplored. In this disclosure, the association of CYP2I2*7 with intrinsic arrhythmia and drug-induced proarrhythmia is reported, which has not been reported before. This association reported in the present disclosure is novel and non-obvious.

[0041] Currently, there is no risk assessment test to verify the proarrhythmic risk of patients in the clinic. Prior to specific surgical and medical interventions, it is important to ascertain the proarrhythmic risk of patients. Genotyping CYP2I2*7 will help diagnose the proarrhythmic risk level of such vulnerable patients so that extra precautions can be taken to mitigate the risk during subsequent intervention (precision diagnosis). Additionally, there are many drugs that are known reversible inhibitors or irreversible inactivators of CYP2J2 (Table 5). Hence, prior to the initiation of these pharmacotherapies, clinicians may genotype the patients for CYP2J2*7 so that the appropriate medications can be prescribed or abstained (precision medicine). Lastly, drug discovery scientists are inventing new candidate entities (NCEs) which might potentially be reversible inhibitors or irreversible inactivators of CYP2J2. In this instance, patients with CYP2J2*7 may be excluded from the clinical studies to avoid drug-induced proarrhythmic risk (safety biomarker). Alternatively, CYP2I2*7 may be associated with a disease state (e.g. coronary artery disease). In this instance, patients with CYP2I2*7 may be selected for the clinical studies to optimize the pharmacotherapeutic outcome (efficacy biomarker).

[0042] Table 5. Reversible inhibitors or irreversible inactivators of CYP2J2

[0043] In one aspect, the present disclosure refers to a method of identifying a subject who has or is predisposed to cardiac arrhythmia, the method comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7;wherein the presence of the allele indicates that the subject has or is predisposed to cardiac arrhythmia.

[0044] As used herein, cardiac arrhythmia, also known as abnormal heart rhythm or irregular heartbeat, refers to any change in the normal sequence of electrical impulses produced by the heart. The abnormal heart rhythms can be slow or fast in rate, having extra beats, or have rhythm irregularity. There are different causes for cardiac arrhythmias, including intrinsic heart problems, endocrinological and metabolic causes. In one example, the cardiac arrhythmia is slow heart rates (less than 60 beats per minute), also called bradycardias. In another example, the cardiac arrhythmia is fast heart rates (more than 100 beats per minute), also called tachycardia. In another example, the cardiac arrhythmia is ventricular tachycardia (VT), which is a very rapid, but regular heartbeat of 100 beats or more a minute occurring in the lower chambers (ventricles) of the heart. In another example, the cardiac arrhythmia is supraventricular tachycardia (SVT), which is a sudden burst of rapid heartbeats that begin and end abruptly, lasting for seconds or hours. In another example, the cardiac arrhythmia is atrial fibrillation (AF), which occurs when there is abnormal electrical activity within the upper chambers (atria) of the heart, resulting in the lower chambers (ventricles) to beat in a fast and irregular manner. In another example, the cardiac arrhythmia is ventricular fibrillation (VF), characterised by very fast and very irregular heartbeats. In one example, the cardiac arrhythmia is intrinsic arrhythmia, or inherent arrhythmia, which is a pre-existing arrhythmia. In one example, intrinsic arrhythmia is inherited arrhythmia that is passed down from generation to generation. Inherited arrhythmia is selected from the group consisting of arrhythmogenic right ventricular dysplasia (ARVD), brugada syndrome (BrS), congenital long QT (LQT), congenital short QT intervals (SQT), early rcpolarization syndrome, and idiopathic ventricular fibrillation (IVF). In another example, intrinsic arrhythmia is established during the life-time of a subject but not inherited from his / her parents. In another example, the cardiac arrhythmia is drug- induced proarrhythmia. Proarrhythmia is defined as the provocation of a new arrhythmia or the aggravation of a pre-existing one during therapy with an antiarrhythmic drug at doses or plasma concentrations below those considered to be toxic. In other words, proarrhythmia is a side effect associated with the administration of some existing antiarrhythmic dings, as well as drugs for other indications. Drug-induced proarrhythmia may include drug-induced bradyarrhythmia, atrial and ventricular proarrhythmia.

[0045] In one example, the drug-induced proarrhythmia may be induced by a CYP2J2 reversible inhibitor or irreversible inactivator. Cytochrome P450 2J2 (CYP2J2), an abundantcardiac epoxygenase, metabolizes endogenous polyunsaturated fatty acid, arachidonic acid (AA), to bioactive regioisomeric epoxyeicosatrienoic acid (EET) metabolites. EETs are lipid mediators with vasodilatory, angiogenic, anti-apoptotic, anti-inflammatory and ion channel regulatory functions, possessing cardioprotective roles. CYP2J2 was reported to be primarily expressed in cardiac tissue and is the most predominantly expressed P450 in human cardiomyocytes, and also in cardiac endothelial cells. As used herein, "CYP2J2 reversible inhibitor" refers to an entity that noncovalently binds to the active and / or allosteric site of CYP2J2 enzyme and reversibly inhibits its ability to metabolize another substrate. With the removal of the entity, the enzymatic activity of CYP2J2 can be restored. As used herein, "CYP2J2 irreversible inactivator" refers to an entity that irrevocably destroys CYP2J2 enzyme or diminishes its activity for example, by covalent modification and can only be restored by de novo protein synthesis. One example of the is a perpetrator drug which is first metabolically activated by the CYP2J2 enzyme into a chemically reactive intermediate that covalently alkylates the P450 apoprotein and / or its prosthetic heme or forms a coordination complex with the heme catalytic ferrous (Fe2+), thereby engendering a distinctive time-dependent loss of its activity via a phenomenon known as mechanism- based inactivation (MBI). The CYP2J2 reversible inhibitor or irreversible inactivator may inhibit and decrease the activity of CYP2J2, e.g., by interfering with the interaction of CYP2J2 with another molecule, e.g., its substrate. The CYP2J2 reversible inhibitor or irreversible inactivator can be selected from the group comprising danazol, dronedarone, amiodarone, bepridil, isradipine, nicardipine, verapamil, lovastatin, simvastatin, ranolazine, quinapril, telmisartan, erlotinib, nilotinib, imatinob, gefitinib, apatinib, motesanib, vatalanib, sorafenib, vanderanib, ibrutinib, osimertinib, infigratinib, cisapride, astcmizolc, loratadine, tcrfcnadinc, ondansetron, domperidone, droperidol, clozapine, haloperidol, pimozide, risperidone, thioridazine, omeprazole, lansoprazole, orphenadrine, clotrimazole, miconazole, ketoconazole, clomiphene, tamoxifen, amodiaquine, mefloquine, ivermectin, albendazole, quercetin, fluoxetine, fluvoxamine, paroxetine methadone, cyclobenzaprine, nortriptyline, budesonide, ritonavir, flunarizine, vandetanib, piperine, mevinolin, LKY-047, and derivatives thereof. This list is non-exhau stive and may include newly approved dings that a e CYP2J2 reversible inhibitors or irreversible inactivators.

[0046] In another example, the drug-induced proarrhythmia may be induced by an antiarrhythmic drug selected from the group consisting of disopyramide, procainamide, quinidine, mexiletine, propafenone, flecainide, d,l-sotalol, amiodarone, dronedarone,bretylium, dofetilide, ibutilide, azimilide, and ajmaline. In another example, the drug-induced proarrhythmia may be induced by an anticancer drug selected from the group consisting of tamoxifen, lapatinib, vandetanib, nilotinib, and arsenic trioxide. In another example, the drug- induced proarrhythmia may be induced by an antifungal drug selected from the group consisting of itraconazole, ketoconazole, fluconazole, and voriconazole. In another example, the drug-induced proarrhythmia may be induced by an antimicrobial drug selected from the group consisting of rrythromycin, clarithromycin, azithromycin, spiramycin, telithromycin, levofloxacin, moxifloxacin, sparfloxacin, gatifloxacin, grepafloxacin, gemifloxacin, ofloxacin, trimcthoprim-sulfamcthoxazolc, pentamidine, quinine, chloroquine, mefloquine, and halofantrine. In another example, the drug-induced proarrhythmia may be induced by an antiviral drug such as foscamct. In another example, the drug-induced proarrhythmia may be induced by an antihistamine selected from the group consisting of astemizole, diphenhydramine, ebastine, terfenadine, and hydroxyzine. In another example, the drug- induced proarrhythmia may be induced by an antidepressant selected from the group consisting of doxepin, venlafaxine, fluoxetine, desipramine, imipramine, clomipramine, paroxetine, sertraline, citalopram, and escitalopram. In another example, the drug-induced proarrhythmia may be induced by an antipsychotic selected from the group consisting of chlorpromazine, prochlorperazine, trifluoperazine, fluphenazine, felbamate, haloperidol, thioridazine, droperidol, mesoridazine, pimozide, risperidone, quetiapine, ziprasidone, lithium, chloral hydrate, pericycline, sertindole, suitopride, zimeldine, maprotiline, and tiapride. In another example, the drug-induced proarrhythmia may be induced by an antimigraine selected from the group consisting of naratriptan, sumatriptan, and zolmitriptan. In another example, the drug- induced proarrhythmia may be induced by a bronchodilator selected from the group consisting of albuterol and salmeterol. In another example, the drug-induced proarrhythmia may be induced by a diuretic selected from the group consisting of indapamide, thiazide, and furosemide. In another example, the drug-induced proarrhythmia may be induced by a gastrointestinal stimulant selected from the group consisting of cisapride, metoclopramide, and domperidone. In another example, the drug-induced proarrhythmia may be induced by a hormone selected from the group consisting of octreotide and vasopressin. In another example, the drug-induced proarrhythmia may be induced by an immunosuppressive such as tacrolimus. In another example, the drug-induced proarrhythmia may be induced by a drug selected from the group consisting of probucol, methadone, cocaine, amantadine, aconitine, veratridine, vincamine, terodiline, budipine, tizanidine, and organophosphorus compounds.

[0047] In order to identify or diagnose a subject who has or is predisposed to cardiac arrhythmia, an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7 is to be tested in nucleic acid from a sample obtained from the subject. As used herein, the term “sample” includes tissues, cells, body fluids and isolates thereof etc., isolated from a subject, as well as tissues, cells and fluids etc. present within a subject Examples of samples include: whole blood, blood fluids (e.g. serum and plasm), lymph and cystic fluids, sputum, stool, tears, mucus, hair, skin, ascitic fluid, cystic fluid, urine, nipple exudates, nipple aspirates, sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, archival samples, explants and primary and / or transformed cell cultures derived from patient tissues etc. The sample may be untreated, treated, diluted or concentrated from a patient.

[0048] In step a of the method as disclosed herein, a sample selected from the group consisting of blood, saliva, or buccal cells is obtained from the subject. The sample can be collected by a tool such as a syringe for drawing blood, a swab for collecting saliva, a buccal swab for collecting buccal cells, or other sample collection devices.

[0049] The sample contains nucleic acid to be tested in step b of the method as disclosed herein. In one example, the nucleic acid is a nucleic acid extract from the sample as disclosed herein. In another example, the method as disclosed herein comprises a further step of preparing the nucleic acid extract from the sample prior to the testing in step b. The nucleic acid such as DNA in saliva originates from cells that are shed from the inner linings of the mouth and from white blood cells. These DNA-containing cells are collected, and the DNA is then extracted by various methods as disclosed herein, for testing in step b. In another example, the nucleic acid is not extract from the sample as disclosed herein and the method as disclosed herein does not comprise a further step of preparing the nucleic acid extract from the sample prior to the testing in step b.

[0050] As used herein, a "nucleic acid extract" or an "isolated nucleic acid" generally is one that contains a SNP CYP2.T2*7 of the present invention or one that hybridizes to such molecule such as a nucleic acid with a complementary sequence, and is separated from most other nucleic acids present in the sample containing the nucleic acid molecule. Moreover, "nucleic acid extract" or an "isolated nucleic acid", such as a cDNA molecule containing a SNP CYP2I2*7 of the present invention, can be substantially free of other cellular material, or culture medium, or other chemicals A nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered "isolated." Further examples of "nucleic acid extract" or an "isolated nucleic acid" include purified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated SNP- containing DNA molecules of the present invention.

[0051] A "nucleic acid extract" or an "isolated nucleic acid" of the present invention further encompasses a SNP- containing polynucleotide that is the product of any one of a variety of nucleic acid amplification methods, which are used to increase the copy numbers of a polynucleotide of interest in a nucleic acid sample. Such amplification methods are well known in the art, and they include but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), linked linear amplification (LLA) and the like, and isothermal amplification methods such as nucleic acid sequence-based amplification (NASBA) and selfsustained sequence replication. As used herein, an "amplified polynucleotide" of the invention is a SNP-containing nucleic acid molecule whose amount has been increased at least two-fold by any nucleic acid amplification method performed in vitro as compared to its starting amount in a test sample. In other preferred embodiments, an amplified polynucleotide is the result of at least ten-fold, fifty-fold, one hundred-fold, one thousand-fold, or even ten thousand-fold increase as compared to its starting amount in a test sample. In a typical PCR amplification, a polynucleotide of interest is often amplified at least fifty thousand-fold in amount over the unamplified genomic DNA, but the precise amount of amplification needed for an assay depends on the sensitivity of the subsequent detection method used.

[0052] The methods and reagents for use in PCR amplification reactions, restriction enzyme digestion and subsequent fragment resolution, and nucleic acid sequencing are well known to those skilled in the ait. In each case, suitable protocols and reagents will largely depend on individual circumstances. Guidance may be obtained from a variety of sources, such as for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992. A person skilled in the art would readily appreciate that various parameters of these procedures may be altered without affecting the ability to achieve the desired product. For example, in the case of PCR amplification, the salt concentration may be varied. Similarly, the amount of DNA used a template may also be varied depending on the amount of DNA available or the optimal amount of template required for efficient amplification.

[0053] A "nucleic acid extract" or an "isolated nucleic acid" of the present invention can be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomicDNA. The nucleic acid, especially DNA, can be double-stranded or single- stranded. Singlestranded nucleic acid can be the coding strand (sense strand) or the complementary non- coding strand (anti- sense strand). DNA, RNA, orPNA segments can be assembled, for example, from fragments of the human genome (in the case of DNA or RNA) or single nucleotides, short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic nucleic acid molecule. In one example, the nucleic acid extract is genomic DNA extracted from whole blood cells of a blood sample obtained from the subject. In another example, the nucleic acid extract is genomic DNA extracted from a saliva sample obtained from the subject. In another example, the nucleic acid extract is genomic DNA extracted from a buccal sample obtained from the subject.

[0054] As used herein, an "allele" refers to a nucleotide at a SNP position (wherein at least two alternative nucleotides exist in the population at the SNP position, in accordance with the inherent definition of a SNP) or may refer to an amino acid residue that is encoded by the codon which contains the SNP position (where the alternative nucleotides that are present in the population at the SNP position form alternative codons that encode different amino acid residues). An "allele" may also be referred to herein as a "variant". Also, an amino acid residue that is encoded by a codon containing a particular SNP may simply be referred to as being encoded by the SNP.

[0055] Those skilled in the art will readily recognize that nucleic acid molecules may be double- stranded molecules and that reference to a particular site on one strand refers, as well, to the corresponding site on a complementary strand. In defining a SNP position, SNP allele, or nucleotide sequence, reference to an adenine, a thymine (uridine), a cytosine, or a guanine at a particular site on one strand of a nucleic acid molecule also defines the thymine (uridine), adenine, guanine, or cytosine (respectively) at the corresponding site on a complementary strand of the nucleic acid molecule. Thus, reference may be made to either strand in order to refer to a particular SNP position, SNP allele, or nucleotide sequence. Probes and primers, may be designed to hybridize to either strand and SNP genotyping methods disclosed herein may generally target either strand.

[0056] References to variant peptides, polypeptides, or proteins of the present invention include peptides, polypeptides, proteins, or fragments thereof, that contain at least one amino acid residue that differs from the corresponding amino acid sequence of the art-known peptide / polypeptide / protein (the art-known protein may be interchangeably referred to as the "wild-type," "reference," or "normal" protein such as wild-type CYP2J2). Such variantpeptides / polypeptides / proteins can result from a codon change caused by a nonsynonymous nucleotide substitution at a protein- coding SNP position (i.e. a missense mutation). Variant peptides / polypeptides / proteins of the present invention can also result from a nonsense mutation (i.e., a SNP that creates a premature stop codon, a SNP that generates a read- through mutation by abolishing a stop codon), or due to any SNP disclosed by the present invention that otherwise alters the structure, function, activity, or expression of a protein, such as a SNP in a regulatory region (e g. a promoter or enhancer) or a SNP that leads to alternative or defective splicing, such as a SNP in an intron or a SNP at an exon / intron boundary. As used herein, CYP2J2*7 is a single nucleotide polymorphism (SNP) involving a G>T substitution in the proximal promoter 50 base pairs (bp) before the transcriptional start site of CYP2J2. This mutation interferes with Spl transcription factor binding, resulting in a reduction in promoter activity, and a reduced translational expression of CYP2J2.

[0057] The primers for use in the methods and kits of the present invention, for genotyping of CYP2J2*7, are typically oligonucleotides of, generally, 15 to 30 bases in length. Such primers can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Not all bases in the primer need reflect the sequence of the template molecule to which the primer will hybridize, the primer need only contain sufficient complementary bases to enable the primer to hybridize to the template. The primer may include additional bases, for example in the form of a restriction enzyme recognition sequence at the 5’ end, to facilitate cloning of the amplified DNA. A primer may also include mismatch bases at one or more positions, being bases that are not complementary to bases in the template, but rather are designed to incorporate changes into the DNA upon base extension or amplification.

[0058] In an example, the genotyping of CYP2I2*7 of the nucleic acid in the sample obtained from the subject as disclosed herein can be done by a method selected from the group consisting of restriction fragment length polymorphism (RFLP), sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, or denaturing gradient gel electrophoresis (DGGE). In one example, genotyping of CYP2J2*7 is efficiently accomplished using a well-established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Specifically, after or without nucleic acid extraction, PCR will then be employed to amplify the targeted sequence using specific primer pairs designed for CYP2J2*7 alleles: 5’- ACTGCTCCTAGCCTGGCC-3’ (SEQ ID NO: 1) and 5’-GGCTCAGACGTCCTC-3’(SEQ IDNO: 2). A person skilled in the art can readily design other primers in any suitable regions 5' and 3' to SNP CYP2J2*7 disclosed herein. Such primers may be used to amplify DNA of any length so long that it contains the SNP of interest (CYP2J2*7) in its sequence. In one example, the PCR products will undergo digestion by a restriction enzyme such as Alul, followed by separation on a 4% agarose gel. The mutant allele will be discerned from the fragments of 117 and 77 base pairs, whereas the wild-type CYP2J2 allele will remain uncut, enabling precise genotyping.

[0059] In another example, the genotyping of CYP2J2*7 of the nucleic acid in the sample obtained from the subject as disclosed herein can be done by an allele- specific method selected from the group consisting of allele -specific probe hybridization, allele-specific primer extension, or allele- specific amplification. For analyzing SNPs, it may be appropriate to use oligonucleotides specific for alternative SNP alleles. Such oligonucleotides that detect single nucleotide variations in target sequences may be referred to by such terms as "allele- specific oligonucleotides," "allele- specific probes," or "allele- specific primers." The design and use of allele-specific probes for analyzing polymorphisms is described in, e.g., Mutation Detection: A Practical Approach, Cotton et ah, eds., Oxford University Press (1998); Saiki et al, Nature 324: 163-166 (1986); Dattagupta, EP235 / 726; and Saiki, WO 89 / 11548.

[0060] The genotyping of CYP2I2*7 of the nucleic acid in the sample obtained from the subject as disclosed herein can be done using a SNP detection reagent, which is an isolated or synthetic DNA or RNA polynucleotide probe or primer or PNA oligomer, or a combination of DNA, RNA and / or PNA, that hybridizes to a segment of a target nucleic acid molecule containing a SNP CYP2J2*7. A detection reagent in the form of a polynucleotide may optionally contain modified base analogues, intercalators or minor groove binders. Multiple detection reagents such as probes may be, for example, affixed to a solid support (e.g. arrays or beads) or supplied in solution (e.g. probe / primer sets for enzymatic reactions such as PCR, RT-PCR, TaqMan assays, or primer-extension reactions) to form a SNP detection kit. A probe or primer typically is a substantially purified oligonucleotide or PNA oligomer. Such oligonucleotide typically comprises a region of complementary nucleotide sequence that hybridizes under stringent conditions to at least about 8, 10, 12, 16, 18, 20, 22, 25, 30, 40, 50, 55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule. Depending on the particular assay, the consecutive nucleotides can either include the target SNP position, or be a specific region in close enough proximity 5' and / or 3' to the SNP position to carry out the desired assay. A primer or probe ofthe present invention is at least about 8 nucleotides in length, at least about 10 nucleotides in length, at least about 12 nucleotides in length, at least about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.

[0061] As used herein, the term "subject" or “patient” refers to patients of human or other mammal and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that “patient” does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (eg. sheep, cows, horses, donkeys, pigs), laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters), companion animals (eg. cats, dogs) and captive wild animals (eg. foxes, deer, dingoes). Preferably, the subject is human.

[0062] A "subject" or “patient” as disclosed herein may be homozygous or heterozygous (carrier) for an allele at the SNP CYP2I2*7 position. SNP CYP2J2*7 is autosomal dominant. Thus, a subject who is heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7 shows reduced expression and activity of CYP2J2. In another example, the subject is wildtype i.e. CYP2J2*1 / *1. Wildtype subject is not homozygous (CYP2J2*7 / *7) nor heterozygous (CYP2J2*l / *7).

[0063] In one example, the subject as disclosed herein did not have cardiac arrhythmia (intrinsic arrhythmia or drug-induced pro arrhythmia) prior to the testing according to the method as disclosed herein. Tn another example, the subject as disclosed herein did have cardiac arrhythmia (intrinsic arrhythmia or drug-induced proarrhythmia) prior to the testing according to the method as disclosed herein.

[0064] Genotyping CYP2J2*7 is applicable to the diagnosis of patients who have a proarrhythmic substrate and potentially susceptible to drug-induced arrhythmic risk (precision diagnosis). In the event that the allele at SNP CYP2J2*7 is not detected, i.e. the subject is wildtype, the subject is diagnosed as not having or predisposed to cardiac arrhythmia (intrinsic arrhythmia or drug-induced proarrhythmia). Then a CYP2.T2 reversible inhibitor or irreversible inactivator as disclosed herein can be administered to the subject to treat or prevent the development of cardiac arrhythmia (which is intrinsic arrhythmia or drug-induced proarrhythmia). In the event that the allele at SNP CYP2J2*7 is indeed detected, i.e. the subject is homozygous or heterozygous (carrier) for an allele at the SNP CYP2J2*7 position, then the subject is diagnosed as having or predisposed to cardiac arrhythmia (which is intrinsic arrhythmia or drug-induced proarrhythmia). In this case, such subject shall not be treated with a CYP2.T2 reversible inhibitor or irreversible inactivator as disclosed herein, or shall be treatedinstead with a CYP2J2 reversible inhibitor or irreversible inactivator at a reduced dose, or optimized dosing regimen (including the dose, dosing frequency, dosage formulation, route of administration, and duration of treatment), such that the ablation of CYP2J2 activity becomes clinically less important. In one example, the dose of the CYP2J2 reversible inhibitor or irreversible inactivator per administration is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In another example, the dosing frequency of the CYP2J2 reversible inhibitor or irreversible inactivator is reduced by 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. In another example, the time interval between administrations of the CYP2J2 reversible inhibitor or irreversible inactivator is increased by 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. In another example, the duration of the treatment of the CYP2J2 reversible inhibitor or irreversible inactivator is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. Alternatively, a different therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator shall be administered to treat or prevent the development of cardiac arrhythmia (which is intrinsic arrhythmia or drug-induced proarrhythmia). As used herein, "the same therapeutic class" refers to drugs that have the same therapeutic objective or treat the same disease as the CYP2.T2 reversible inhibitor or irreversible inactivator which shall not be used or shall be used at a reduced dose or optimized dosing regimen when the allele at SNP CYP2J2*7 is indeed detected. In one example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as amiodarone and dronedarone which arc anti-atrial fibrillation drugs shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. anti-atrial fibrillation drugs, which can be used as alternative therapy may be beta-blockers such as atenolol and metoprolol. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as isradipine, nicardipine, and verapamil which axe calcium-channel blocker shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. calcium-channel blocker, which can be used as alternative therapy may be amlodipine and nifedipine. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygousCYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as lovastatin, mevinolin and simvastatin which are cholesterol-lowering drugs shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. cholesterol-lowering drugs, which can be used as alternative therapy may be atorvastatin and rosuvastatin. Tn another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as ranolazine which is an anti-anginal drug shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.c. anti-anginal drug, which can be used as alternative therapy may be isosorbide mononitrate, and glyceryl trinitrate sublingual. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as quinapril which is an Angiotensin-converting Enzyme Inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agents from the same therapeutic class, i.e. Angiotensin-converting Enzyme Inhibitor, which can be used as alternative therapy may be enalapril, captopril and lisinopril. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as telmisartan which is an Angiotensin Receptor Blocker shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Angiotensin Receptor Blocker, which can be used as alternative therapy may be losartan and valsartan. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as erlotinib, nilotinib, imatinib, and gefitinib which are Tyrosine Kinase Inhibitors shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Tyrosine Kinase Inhibitors, which can be used as alternative therapy may be sunitinib and dasatinib. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as apatinib, motesanib, vatalanib, sorafenib, and vandetanib which are Multikinase Inhibitors shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Multikinase Inhibitors, which can be used as alternative therapy may be pazopanib and lenvatinib. In another example, when theallele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as vandetanib which is a Protein Kinase Inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Protein Kinase Inhibitor, which can be used as alternative therapy may be vemurafenib. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as ibrutinib which is a Bruton's Tyrosine Kinase Inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Bruton's Tyrosine Kinase Inhibitor, which can be used as alternative therapy may be acalabrutinib and zanubrutinib. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as osimertinib which is an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, which can be used as alternative therapy may be afatinib. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2.T2 reversible inhibitor or irreversible inactivator such as infigratinib which is a Fibroblast Growth Factor Receptor- specific Tyrosine Kinase Inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Fibroblast Growth Factor Receptor- specific Tyrosine Kinase Inhibitor, which can be used as alternative therapy may be pcmbrolizumab. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as cisapride which is a gastroprokinetic shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. gastroprokinetic, which can be used as alternative therapy may be metoclopramide and erythromycin. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as astemizole, loratadine and terfenadine which are antihistamine drugs for treating allergic conditions shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the sametherapeutic class, i.e. antihistamine drugs, which can be used as alternative therapy may be cetirizine and chlorpheniramine. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as ondansetron which is a serotonin 5-HT3 receptor antagonist shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. serotonin 5-HT3 receptor antagonist, which can be used as alternative therapy may be transdermal scopolamine and granisetron. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as domperidone and droperidol which are dopamine receptor antagonists shall not be used or shah be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. dopamine receptor antagonists, which can be used as alternative therapy may be metoclopramide. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as clozapine, haloperidol, pimozide, risperidone, and thioridazine which are antipsychotic shall not be used or shah be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. antipsychotic, which can be used as alternative therapy may be quetiapine, olanzapine, and aripiprazole. Tn another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as omeprazole, and lansoprazole which are Proton Pump Inhibitors shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Proton Pump Inhibitors, which can be used as alternative therapy may be pantoprazole. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*1 / *7 or homozygous CYP2J2*7 / *7), the CYP2.T2 reversible inhibitor or irreversible inactivator such as orphenadrine which is an anticholinergic shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. anticholinergic, which can be used as alternative therapy may be hyoscine butylbromide. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as danazol which is an androgenic shall not be used or shall be used at a reduced dose or optimized dosing regimen. The differenttherapeutic agent from the same therapeutic class, i.e. androgenic, which can be used as alternative therapy may be depot leuprolide. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as clotrimazole, miconazole, and ketoconazole which are anti-fungal drugs shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. anti-fungal drug, which can be used as alternative therapy may be fluconazole, itraconazole, and tinidazole. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2I2 reversible inhibitor or irreversible inactivator such as clomiphene and tamoxifen which are Selective Estrogen Receptor Modulators shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Selective Estrogen Receptor Modulators, which can be used as alternative therapy may be raloxifene. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2I2*l / *7 or homozygous CYP2I2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as amodiaquine and mefloquine which are antimalarial drugs shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. antimalarial drugs, which can be used as alternative therapy may be chloroquine, malarone, and primaquine. Tn another example, when the allele at SNP CYP2I2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2I2 reversible inhibitor or irreversible inactivator such as ivermectin and albendazole which are antihelmintic drugs shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. antihelmintic drugs, which can be used as alternative therapy may be praziquantel and mebendazole. In another example, when the allele at SNP CYP2I2*7 is detected (whether heterozygous CYP2J2*1 / *7 or homozygous CYP2J2*7 / *7), the CYP2.T2 reversible inhibitor or irreversible inactivator such as quercetin which is a flavonoid shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. flavonoid, which can be used as alternative therapy may be rutin. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as fluoxetine, fluvoxamine, and paroxetine which are Selective Serotonin- reuptake Inhibitors shall not be used or shall be used at a reduced dose or optimized dosingregimen. The different therapeutic agent from the same therapeutic class, i.e. Selective Serotonin-reuptake Inhibitors, which can be used as alternative therapy may be sertraline, fluoxetine, and escitalopram. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as methadone which is an analgesic shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. analgesic, which can be used as alternative therapy may be ketamine. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as cyclobenzaprine and notriptyline which are antidepressants shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. antidepressants, which can be used as alternative therapy may be amitriptyline, and sertraline. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as budesonide which is a corticosteroid shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. corticosteroid, which can be used as alternative therapy may be betamethasone and hydrocortisone. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as ritonavir which is a Protease Inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Protease Inhibitor, which can be used as alternative therapy may be amprenavir and atazanavir. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2* l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as flunarizine which is a Calcium Antagonist shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. Calcium Antagonist, which can be used as alternative therapy may be cinnarizine. In another example, when the allele at SNP CYP2J2*7 is detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), the CYP2J2 reversible inhibitor or irreversible inactivator such as piperine which is an alkaloid, a NF-kappaB inhibitor shall not be used or shall be used at a reduced dose or optimized dosing regimen. The different therapeutic agent from the same therapeutic class, i.e. an alkaloid, a NF-kappaB inhibitor,which can be used as alternative therapy may be lobeline. The exemplar}' different therapeutic agent from the same therapeutic class to be used is shown in Table 6. A person skilled in the art would appreciate that this list of "different therapeutic agent from the same therapeutic class" is not exhaustive. As long as a CYP2J2 reversible inhibitor or irreversible inactivator is not suitable to be administered or shall be administered at a reduced dose or optimized dosing regimen to a subject who is homozygous or heterozygous (carrier) for an allele at the SNP CYP2J2*7 position, a different therapeutic agent with same therapeutic objective or treat the same disease as the CYP2J2 reversible inhibitor or irreversible inactivator can be used, which may include existing and newly developed drugs.

[0065] Table 6. Exemplary different therapeutic agent from the same therapeutic class to be used when the allele at SNP CYP2J2*7 is detected

[0066] As used herein, the terms "drug" and "therapeutic agent" are used interchangeably, and may include, but are not limited to, small molecule compounds, biologies (e.g., antibodies, proteins, protein fragments, fusion proteins, glycoproteins, etc.), nucleic acid agents (e.g., antisense, RNAi / siRNA, and microRNA molecules, etc ), vaccines, etc., which may be used for therapeutic and / or preventive treatment of a disease (e.g. cardiac arrhythmia such as intrinsic arrhythmia or drug-induced proarrhythmia). In one example, the therapeutic agent is from the same therapeutic class as a CYP2J2 reversible inhibitor or irreversible inactivator but is not a CYP2J2 reversible inhibitor or irreversible inactivator, as disclosed herein (Table 6).

[0067] The CYP2J2 reversible inhibitor or irreversible inactivator or the therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator can be administered by standard routes. In general, therapeutic agents will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal, or by suppository), or parenteral (e.g., intramuscular, intravenous, or subcutaneous) administration. The preferred manner of administration is oral or parenteral using a convenient daily dosage regimen, which can be adjusted according to the degree of affliction. Oral compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions.

[0068] The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills, or capsules are preferred) and the bioavailability of the drug substance.

[0069] The CYP2J2 reversible inhibitor or irreversible inactivator or the therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator can be administered together with a pharmaceutically acceptable carrier, diluent, and adjuvant, in a "therapeutically efficacious dose”. The carriers, diluents and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxancs, such as methyl polysiloxanc, phenyl polysiloxanc and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 -butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0070] As used herein the term "therapeutically efficacious dose” includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “efficacious dose”. However, for any given case, an appropriate “efficacious dose” may be determined by one of ordinary skill in the ait using only routine experimentation.

[0071] Therapeutically efficacious doses of therapeutic agents may range from, for example, approximately 0.01-50 mg per kilogram body weight of the recipient per day; preferably about 0.1-20 mg / kg / day. Thus, as an example, for administration to a 70 kg person, the dosage range would most preferably be about 7 mg to 1.4 g per day.

[0072] In another aspect, the present disclosure refers to a method of treating or preventing cardiac arrhythmia in a subject having or predisposed to cardiac arrhythmia, comprising: a. obtaining a sample from the subject; b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein when the allele is present, the method further comprising administering a reduced dose or optimizing the dosing regimen of a CYP2J2 reversible inhibitor or irreversible inactivator compared to when the allele is absent, or administering a therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator to the subject.

[0073] As used herein the term "treat", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

[0074] Genotyping CYP2J2*7 is applicable to the optimization of pharmacotherapy of regulatory approved drugs that are CYP2J2 inhibitors or inactivators where CYP2J2*7 carriers are contraindicated (precision medicine). In the event that the allele at SNP CYP2J2*7 is not detected, then a CYP2J2 reversible inhibitor or irreversible inactivator as disclosed herein can be administered to the subject to treat or prevent the development of cardiac arrhythmia (which is intrinsic arrhythmia or drug-induced proarrhythmia). In the event that the allele at SNP CYP2J2*7 is indeed detected (whether heterozygous CYP2J2*l / *7 or homozygous CYP2J2*7 / *7), then a CYP2J2 reversible inhibitor or irreversible inactivator as disclosedherein should not be administered to the subject, or a reduced dose of the CYP2J2 reversible inhibitor or irreversible inactivator, or optimized dosing regimen shall be adopted, or a different therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator shall be administered to treat or prevent the development of cardiac arrhythmia (which is intrinsic arrhythmia or drug-induced proarrhythmia).

[0075] In another aspect, the present disclosure refers to a method for establishing a prognosis for a patient having or predisposed to cardiac arrhythmia and treated by a CYP2J2 reversible inhibitor or irreversible inactivator, comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein the presence of the allele indicates poor prognosis.

[0076] The SNP CYP2J2*7 of the present invention may have utility in determining why certain previously developed drugs such as a CYP2J2 reversible inhibitor or irreversible inactivator performed poorly in clinical trials and may help identify other drugs that this particular patient population having SNP CYP2J2*7 can benefit from, such as a therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator, as disclosed herein.

[0077] Tn another aspect, the present disclosure refers to a detection reagent for carrying out the method as disclosed herein, wherein said detection reagent is an allele- specific probe or an allele-specific primer.

[0078] For analysing SNPs, it may be appropriate to use oligonucleotides specific for alternative SNP alleles. Such oligonucleotides that detect single nucleotide variations in target sequences may be referred to by such terms as "allele- specific oligonucleotides," "allelespecific probes," or "allele- specific primers." The design and use of allele-specific probes for analyzing polymorphisms is described in, e g., Mutation Detection: A Practical Approach, Cotton et ah, eds., Oxford University Press (1998); Saiki et al, Nature 324: 163-166 (1986); Dattagupta, EP235 / 726; and Saiki, WO 89 / 11548. While the design of each allele-specific primer or probe depends on variables such as the precise composition of the nucleotide sequences flanking a SNP position in a target nucleic acid molecule, and the length of the primer or probe, another factor in the use of primers and probes is the stringency of the condition under which the hybridization between the probe or primer and the target sequence is performed. Higher stringency conditions utilize buffers with lower ionic strength and / or a higher reactiontemperature, and tend to require a more perfect match between probe / primer and a target sequence in order to form a stable duplex. If the stringency is too high, however, hybridization may not occur at all. In contrast, lower stringency conditions utilize buffers with higher ionic strength and / or a lower reaction temperature, and permit the formation of stable duplexes with more mismatched bases between a probe / primer and a target sequence.

[0079] In a hybridization-based assay, allele- specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms (e.g., alternative SNP alleles / nucleotides) in the respective DNA segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant detectable difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles or significantly more strongly to one allele. While a probe may be designed to hybridize to a target sequence that contains a SNP site such that the SNP site aligns anywhere along the sequence of the probe, the probe is preferably designed to hybridize to a segment of the target sequence such that the SNP site aligns with a central position of the probe (e.g., a position within the probe that is at least three nucleotides from either end of the probe). This design of probe generally achieves good discrimination in hybridization between different allelic forms. In another embodiment, a probe or primer may be designed to hybridize to a segment of target DNA such that the SNP aligns with either the 5' most end or the 3' most end of the probe or primer.

[0080] Oligonucleotide probes and primers may be prepared by methods well known in the art. Chemical synthetic methods include, but are not limited to, the phosphotriester method described by Narang et ah, Methods in Enzymology 68:90 (1979); the phosphodi ester method described by Brown et ah, Methods in Enzymology 68: 109 (1979); the diethylphosphoamidate method described by Beaucage et ah, Tetrahedron Letters 22: 1859 (1981), and the solid support method described in U.S. Patent No. 4,458,066.

[0081] In another aspect, the present disclosure refers to a test kit comprising one or more containers containing the detection reagent as disclosed herein and one or more components selected from the group consisting of an enzyme, polymerase enzyme, ligase enzyme, buffer, amplification primer pair, dNTPs, ddNTPs, positive control nucleic acid, negative control, nucleic acid extraction reagent, and instructions for using said test kit which instruct that the presence of the CYP2J2*7 allele.

[0082] The term "kit" as used herein in the context of SNP detection reagents, are intended to refer to such things as combinations of multiple SNP detection reagents, or one or more SNP detection reagents in combination with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as packaging intended for commercial sale, substrates to which SNP detection reagents are attached, electronic hardware components, etc.). Accordingly, the present invention further provides SNP detection kits and systems, including but not limited to, packaged probe and primer sets, arrays / microarrays of nucleic acid molecules, and beads that contain one or more probes, primers, or other detection reagents for detecting one or more SNPs of the present invention. The kits / systems can optionally include various electronic hardware components; for example, arrays ("DNA chips") and microfluidic systems ("lab-on-a-chip" systems) provided by various manufacturers typically comprise hardware components. Other kits / systems (e.g., probe / primer sets) may not include electronic hardware components, but may be comprised of, for example, one or more SNP detection reagents (along with, optionally, other biochemical reagents) packaged in one or more containers.

[0083] In some embodiments, a SNP detection kit typically contains one or more detection reagents and other components (e.g. a buffer, enzymes such as DNA polymerases or ligases, chain extension nucleotides such as deoxynucleotide triphosphates, and in the case of Sanger- type DNA sequencing reactions, chain terminating nucleotides, positive control sequences, negative control sequences, and the like) necessary to carry out an assay or reaction, such as amplification and / or detection of a SNP-containing nucleic acid molecule. A kit may further contain means for determining the amount of a target nucleic acid, and means for comparing the amount with a standard, and can comprise instructions for using the kit to detect the SNP- containing nucleic acid molecule of interest. In one embodiment of the present invention, kits are provided which contain the necessary reagents to carry out one or more assays to detect one or more SNPs disclosed herein In a preferred embodiment of the present invention, SNP detection kits / systems are in the form of nucleic acid arrays, or compartmentalized kits, including microfluidic / lab-on-a-chip systems. Exemplary kits of the invention can comprise a container containing a SNP detection reagent which detects a SNP disclosed herein, said container can optionally be enclosed in a package (e g., a box for commercial sale), and said package can further include other containers containing any or all of the following: enzyme (e.g., polymerase or ligase, any of which can be thermostable), dNTPs and / or ddNTPs (which can optionally be detectably labeled, such as with a fluorescent label or mass tag, and such labelcan optionally differ between any of the dATPs, dCTPs, dGTPs, dTTPs, ddATPs, ddCTPs, ddGTPs, and / or ddTTPs, so that each of these dNTPs and / or ddNTPs can be distinguished from each other by detection of the label, and any of these dNTPs and / or ddNTPs can optionally be stored in the same container or each in separate containers), buffer, controls (e.g., positive control nucleic acid, or a negative control), reagent(s) for extracting nucleic acid from a test sample, and instructions for using the kit (such as instructions for correlating the presence or absence of a particular allele or genotype with an increased or decreased risk for the disease). The SNP detection reagent can comprise, for example, at least one primer and / or probe, any of which can optionally be allele- specific, and any of which can optionally be detectably labeled (e g., with a fluorescent label).

[0084] In certain embodiments of the present invention, a kit may further contain tools for collecting a sample (e.g., buccal cells, saliva, blood, etc.) from the subject as disclosed herein (e.g., a syringe for drawing blood, a swab for collecting saliva, a buccal swab for collecting buccal cells, or other sample collection device). The sample is then sent to a laboratory or other facility that tests the sample for SNP CYP2J2*7 disclosed herein (e.g. to determine the genotype of SNP CYP2J2*7 disclosed herein, such as to determine the patient's predicted response to CYP2J2 reversible inhibitor or irreversible inactivator treatment and risk for developing cardiac arrhythmia, and the results of the test (e.g. the patient' s genotype at SNP CYP2J2*7 and / or the patient' s predicted response to CYP2J2 reversible inhibitor or irreversible inactivator treatment, or risk for developing cardiac arrhythmia based on their SNP genotype) are provided back to the medical practitioner (and / or directly to the patient and / or to another party such as a hospital, medical insurance company, genetic counselor, etc.) who may then provide or otherwise convey the results to the patient. The results are typically provided in the form of a report, such as described above.

[0085] Certain exemplary embodiments of the invention provide the following compositions and uses: (1) a reagent (such as an allele- specific probe or primer, or any other oligonucleotide or other reagent suitable for detecting a polymorphism disclosed herein, which can include detection of any allele of the polymorphism) for use as a diagnostic or predictive agent for determining cardiac arrhythmia including intrinsic arrhythmia and drug-induced proarrhythmia; (2) a kit, device, array, or assay component that includes or is coupled with the reagent of (1) above for use in determining cardiac arrhythmia including intrinsic arrhythmia and drug-induced proarrhythmia; (3) the use of the reagent of (1) above for the manufacture of a kit, device, array, or assay component for determining cardiac arrhythmia including intrinsicarrhythmia and drug-induced proarrhythmia; and (4) the use of a polymorphism CYP2J2*7 disclosed herein for the manufacture of a reagent for use as a diagnostic or predictive agent for determining cardiac arrhythmia including intrinsic arrhythmia and drug-induced proarrhythmia.

[0086] The various methods described herein, such as correlating the presence or absence of a polymorphism CYP2J2*7 with the possibility of a subject as having or predisposed to cardiac arrhythmia, or predicted response of an individual to a drug such as a C YP2J2 reversible inhibitor or irreversible inactivator, particularly for reducing the risk for drug-induced proarrhythmia, can be carried out by automated methods such as by using a computer (or other apparatus / devices such as biomedical devices, laboratory instrumentation, or other apparatus / devices having a computer processor) programmed to carry out any of the methods described herein. For example, computer software (which may be interchangeably referred to herein as a computer program) can perform the step of correlating the presence or absence of a polymorphism CYP2J2*7 in an individual with an altered (e g., increased or decreased) response (or no altered response) to CYP2J2 reversible inhibitor or irreversible inactivator, particularly for reducing the risk for cardiac arrhythmia. Accordingly, certain embodiments of the invention provide a computer (or other apparatus / device) programmed to carry out any of the methods described herein.

[0087] The results of a test (e.g., an individual's predicted responsiveness to CYP2J2 reversible inhibitor or irreversible inactivator treatment or other treatments, or an individual's risk for developing cardiac arrhythmia, based on assaying an individual's allele(s) / genotype at SNP CYP2J2*7 disclosed herein, and / or any other information pertaining to a test, may be referred to herein as a "report" . A tangible report can optionally be generated as part of a testing process (which may be interchangeably referred to herein as "reporting", or as "providing" a report, "producing" a report, or "generating" a report). Examples of tangible reports may include, but are not limited to, reports in paper (such as computer-generated printouts of test results) or equivalent formats and reports stored on computer readable medium (such as a CD, USB flash drive or other removable storage device, computer hard drive, or computer network server, etc ). Reports, particularly those stored on computer readable medium, can be part of a database, which may optionally be accessible via the internet (such as a database of patient records or genetic information stored on a computer network server, which may be a "secure database" that has security features that limit access to the report, such as to allow only the patient and the patient's medical practitioners to view the report while preventing otherunauthorized individuals from viewing the report, for example). In addition to, or as an alternative to, generating a tangible report, reports can also be displayed on a computer screen (or the display of another electronic device or instrument). A report can further be "transmitted" or "communicated" such as to the individual who was tested, a medical practitioner (e g., a doctor, nurse, clinical laboratory practitioner, genetic counsellor, etc ), a healthcare organization, a clinical laboratory, and / or any other party or requester intended to view or possess the report. The act of "transmitting" or "communicating" a report can be by any means known in the art, based on the format of the report. Furthermore, "transmitting" or "communicating" a report can include delivering / sending a report ("pushing") and / or retrieving ("pulling") a report. For example, reports can be transmitted / communicated by various means, including being physically transferred between parties (such as for reports in paper format) such as by being physically delivered from one party to another, or by being transmitted electronically or in signal form (e.g., via e-mail or over the internet, by facsimile, and / or by any wired or wireless communication methods known in the art) such as by being retrieved from a database stored on a computer network server, etc.

[0088] In certain exemplary embodiments, the invention provides computers (or other apparatus / devices such as biomedical devices or laboratory instrumentation) programmed to carry out the methods described herein. For example, in certain embodiments, the invention provides a computer programmed to receive (i.e., as input) the identity (e g., the allele(s) or genotype at SNP CYP2J2*7) and provide (i.e., as output) the predicted drug responsiveness or disease risk. Such output (e g., communication of disease risk, disease diagnosis or prognosis, drug responsiveness, etc.) may be, for example, in the form of a report on computer readable medium, printed in paper form, and / or displayed on a computer screen or other display.

[0089] For the methods as disclosed herein, they may further comprise step of correlating the CYP2J2*7 genotype with electrocardiogram (ECG) data obtained from an appropriate human database. Given that the allele frequency of CYP2J2*7 is 0.054 (5.4%) based on the SGI OK data, a sufficient sample size of heterozygous and homozygous CYP2J2*7 carriers should exist within the appropriate human database.

[0090] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer” includes a plurality of primers, including mixtures and combinations thereof.

[0091] As used herein, the term “comprising” means “including.” Variations of the word "comprising", such as “comprise” and “comprises,” have correspondingly varied meanings.Thus, for example, a composition “comprising” X may consist exclusively of X or may include one or more additional unrecited components.

[0092] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1 % of the stated value, or + / - 0.5% of the stated value.

[0093] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0094] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0095] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0096] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0097] Other embodiments are within the following claims and non-limiting examples.EXAMPLES

[0098] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0099] In this study, novel human embryonic stem cell-derived cardiomyocyte (hESC-CM) genotype models were utilized to investigate if the SNP results in predisposition to both intrinsic arrhythmia and drug-induced proarrhythmia. Firstly, a differentiation protocol was successfully developed referencing the original GiWi methodology and it was demonstrated that CYP2J2 transcriptional expression was reduced in CYP2J2*7-homozygous (HM) hESC- CMs relative to wild-type (WT) controls. Next, utilizing fluorescent probe ER-BnXPI (synthesis route depicted in Figure 9, characterization depicted in Figures 10-12) as a specific probe substrate, it was reported that surrogate Vmax of ER-BnXPI demethylation was diminished in HM hESC-CMs, with apparent Kmremaining unaltered. Lastly, it was showed that HM hESC-CMs exhibited substantially shorter and more variable basal beat-to-beat (BTB) intervals, and were subject to more significant alterations in mean and variability of BTB intervals upon exposure to known CYP2J2 inhibitor danazol, indicating an elevated risk of inherent and drug-induced proarrhythmia. It was also found that both CYP2J2*l / *7 and CYP2J2*7 / *7 hESC-CMs exhibited reduced transcriptional expression, catalytic activities and heighted intrinsic and drug-induced proarrhythmic risks compared to CYP2I2 WT hESC-CMs. Hence, the findings strongly implicate C YP212*7 as a predisposing factor to cardiac arrhythmia and CYP2.T2-reversible inhibitor or irreversible inactivator induced proarrhythmia.

[0100] Example 1 - Materials and Methods

[0101] Chemicals

[0102] Reagents were all analytical grade and above except for the fluorescent probe ER- BnXPI. Chemicals were all procured from Sigma-Aldrich (US), except oleic acid (Cayman Chemical, US) and HPLC-grade acetonitrile (ACN) (Tedia Company, US).

[0103] Cell Culture

[0104] Siem Cell Culture- GCaMP6s-expressing WT and HM H7 hESCs were graciously provided by Dr Soh Boon Seng (Figure 8) and maintained in a 37°C, 5% CO? incubator on Matrigel -coated (Coming, US) 6-well plates containing StemMACs iPS-Brew XF (StemMACs) (Miltenyi Biotec, Germany). hESCs were passaged with 1 mg / mL Collagenase IV (ThermoFisher Scientific, US) upon attaining 80-90% confluency.

[0105] Stem Cell Differentiation- The GiWi protocol was referenced and adapted for cardiomyocyte differentiation [58,74,75], hESCs were first dissociated into singular cells with Accutase (ThermoFisher) and reseeded onto fresh Matrigel-coated 6-well plates. Once fully confluent, differentiation was initiated on Day 0 (DO) by treating hESCs with 12 pM of Wnt activator CHIR99021 (CHIR) (STEMCELL Technologies, Canada) in RPMI-1640 (Cytiva, UK) supplemented with insulin-free B27 (ThermoFisher) and 1% penicillin-streptomycin (ThermoFisher) (RPMI B-). This was followed by a reduced 3 pM CHIR maintenance dose on DI, and 5 pM of Wnt inhibitor IWP-2 (Miltenyi Biotec) on D3. On D5, medium was refreshed with RPMI B-, and subsequently RPMI-1640 with insulin-supplemented B27 (ThermoFisher) and 1% penicillin-streptomycin (RPMI B+) on D7, after which fresh RPMI B+ was introduced every 3 days. On DI 9, medium was swapped to glucose-free RPMI-1640 (Cytiva) containing insulin-supplemented B27, 10 mM galactose, 100 pM oleic acid and 50 pM palmitic acid to facilitate cardiomyocyte selection and metabolic maturation via promoting fatty acid |3- oxidation [58,76],

[0106] Flow Cytometry

[0107] To evaluate differentiation efficiency, hESC-CMs between D25-50 of differentiation were harvested with 0.25% Trypsin-EDTA (ThermoFisher) and immunolabelled with 0.5% v / v anti-human CD172a / p (SIRPa / p) conjugated antibodies (BioLegend, US) in PBS (Cytiva) containing 1% FBS (Cytiva) for 30 min in a 37°C, 5% CO2 incubator. Stained hESC-CMs were washed thrice and resuspended in PBS, and subsequently subjected to flow cytometric analysis with a CytoFLEX-LX flow cytometer (Beckman Coulter, US) The excitation and emission wavelengths were 498 nm and 517 nm respectively.

[0108] Cell Viability Assay

[0109] hESC-CMs between D11-D15 of differentiation were reseeded in Matrigel-coated 96-well plates with a cellular density of 4.0xl04cells / well, and treated with varying concentrations of danazol (0.01-20 pM) or ER-BnXPI fluorescent probe (0.01-25 pM) for 24 h in a 37°C, 5% CO2 incubator. CellTiter-Glo Kit (Promega, US) was employed to assess cell viability following manufacturer protocols, with resultant luminescence evaluated utilizing anInfinite 200 microplate reader (Tecan, Switzerland). ICso values, indicating the maximum non- cytotoxic concentration, were derived from the nonlinear regression curve (log(inhibitor) vs. response, 4 parameters) generated by GraphPad Prism 8.0.2 (Dotmatics, US). Cell viability data of astemizole is shown in Figure 13.[001 10] RNA extraction and quantification, Reverse Transcription and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis

[0111] Total RNA was isolated and purified from hESC-CMs between D25-30 of differentiation with ReliaPrep Minicolumns (Promega) following manufacturer protocols. Qualitative and quantitative analyses were performed using a Nanodrop One Spectrophotometer (ThermoFisher). 1 pg of RNA was denatured and cleared of genomic DNA (gDNA) via incubation with gDNA Wiper Mix (Vazyme, China) (42°C, 2 min), prior to reverse transcription to complementary DNA (cDNA) with HiScript III RT Supermix (Vazyme). Cycling conditions for reverse transcription involved initial extension (37°C, 15 min) and proceeding inactivation (85°C, 5 s). Subsequently, RT-qPCR analysis was performed in triplicates for each cDNA sample with a total volume of 20 pL per reaction, comprising 4 pL of diluted cDNA (400 ng / pL), 10 pL of I Go'laq qPCR Master Mix (Promega), 5.2 pL of nuclease-free water and 0.4 pL each of the corresponding forward and reverse primers (0.2 pM) (Table 7). Amplification was achieved with a CFX96 Touch thermal cycler (BioRad, US), referencing the following program: pre-incubation (95°C, 120 s), followed by 45 cycles of denaturation (95°C, 5 s), annealing (60°C, 15 s) and extension (72°C, 15 s). Transcriptional analysis was performed referencing the AACt methodology with glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) as endogenous control.

[0112] Table 7: Summary of primers utilized for RT-qPCRSEQ ID NO: 3SEQ ID NO: 4SEQ ID NO: 5SEQ ID:NO: 6SEQ ID NO; 7SEQ ID NO: 8SEQ ID NO: 9SEQ ID NO: IDSEQ ID NO: 11SEQ ID NO: 12

[0113] Fluorescent Probe Assays[001 14] The specific fluorescent probe substrate of CYP2J2, ER-BnXPI, was generously synthesized by Dr Tian Chong (Figures 9-12) referencing the original synthesis by Tian et al.

[0077] , ER-BnXPI was utilized for fluorescent imaging and quantification of CYP2J2 activity via flow cytometry.

[0115] Fluorescence Imaging[001 16] Contracting hESC-CMs between D25-30 of differentiation were incubated with 0.9 pM ER-BnXPI for 30 min in a 37°C, 5% CO2 incubator. Stained hESC-CMs were washed with 1 mL PBS thrice, prior to fluorescent imaging with a Nikon ECLIPSE Ti-S fluorescent microscope (Nikon, US). The excitation and emission wavelengths were 633 nm and 690-750 nm respectively.

[0117] CYP2J2 Activity Assays

[0118] Flow cytometry was employed to characterize CYP2J2 activity in hESC-CMs by measuring the average cellular fluorescent signal upon CYP2J2-mediated ER-BnXPI demethylation. The same staining and processing methodologies were employed as per Section 2.2.3, except that anti-human CD172a / [3 antibodies were substituted with varying concentrations of ER-BnXPI (0.05-25 pM). Flow cytometric analysis was carried out using aCytoFLEX-LX flow cytometer with excitation and emission wavelengths of 633 nm and 690- 750 nm respectively. Emission fluorescence was plotted against ER-BnXPI concentration and fitted in a Michaelis-Menten model using GraphPad Prism 8.0.2 to derive Michaelis-Menten parameters (Kmand Vmax) of ER-BnXPI demethylation.[001 19] Cardio-electrophysiology and Heat-To-Heat (HTH) Assays

[0120] Contracting hESC-CMs between D25-30 of differentiation were pre-incubated with 0.1% DMSO or 10 pM CYP2J2 reversible inhibitor danazol [6,42,47,50] (Figure 14) for 24 h within a 37°C, 5% CO2 incubator. Transient calcium currents in the GCaMP6s-expressing hESC-CMs were imaged before and after drug treatment using a Nikon ECLIPSE Ti-S fluorescent microscope (Nikon) at 20x magnification using excitation and emission wavelengths of 496 nm and 513 nm respectively, and recorded with an Andor Zyla 4.2 sCMOS camera for 1 min at 30 frames-per-second (fps) (EINST Technology, Singapore). Video data was subsequently analyzed with Nikon NIS-Elements AR (Nikon) aided by RStudio v2023.12. 1+402 to pinpoint isolated hESC-CMs as regions-of-interest (RO1) and track changes in fluorescent emissions corresponding to individual contractions.

[0121] Study population

[0122] The study cohort was derived from the Singapore Heart Failure Outcomes and Phenotypes (SHOP) study, with the source dataset including both electronic health records (EHR) and genomic data stored in the BioMed Data Access Repository (BioMedDAR). All participants provided informed consent, and the study protocol was approved by the institutional ethics review boards [CIRB 2015-2194, CIRB 2015-2787, CIRB 2019-2046, DSRB 2013 / 00105]. A total of 1,386 individuals with both EHR and genomic data were included in the final analysis.

[0123] Diplotype calling for CYP2J2 polymorphisms

[0124] Variant call format (VCF) files, previously curated using the GenomeAnalysisToolKit (GATK, v4.0.6.0) best practices workflow, were obtained from BioMedDAR in alignment with the Research Collaboration Agreement (RCA). The VCF files were subsequently truncated to retain only variants on chromosome 1, where the CYP2J2 gene is located. Diplotype calling was performed using ALDY 4.0 to assign CYP2J2 genotypes for further analysis.

[0125] Statistical analysis

[0126] All statistical analysis was performed using R version 4.1.0 with the tidy verse and ggpubr packages. Group comparisons of categorical variables were performed using Fisher’sexact test. To correct for multiple hypothesis testing, Bonferroni correction was applied for p- value adjustments.

[0127] Example 2 - Results: hESC-CM differentiation protocol consistently demonstrated excellent efficiency for both hESC-CM cell lines

[0128] To investigate the impact of the CYP2J2*7 SNP on transcriptional expression, enzymatic activity and cardio-electrophysiology, H7 hESCs were first genetically engineered to express the fluorescent Ca2+-channel reporter GCaMP6s to enable visualization of calcium transients. After affirming wild-type CYP2J2 homozygosity in WT hESCs via genetic sequencing (Figure 8A), CRISPR-directed mutagenesis was employed to generate EIM genotype models (Figure 2A), whereby successful transformation was confirmed by further genotyping (Figure 8B). Both cell lines were successfully subjected to directed differentiation into cardiomyocytes following an optimized protocol (Figure 2B) Flow cytometric analysis revealed that the average purity of both WT and HM hESC-CM batches exceeded 90% by D25 of the differentiation timeline, evidenced by their expression of the cardiac marker signal regulatory protein-alpha (SIRPa) (Figure 2C and 2D). Spontaneously contracting cardiomyocytes were consistently observed between D9-12, although such a phenomenon was also noted as early as D7 and as late as DI 6. Additionally, both cell lines exhibited high constitutive transcriptional expression of cardiac marker cardiac troponin 2 (TNNT2) (Figure 2E), further testifying the protocol’s effectiveness.

[0129] Example 3 - Results: CYP2J2 was the predominant P450 epoxygenase in hESC- CMs and demonstrated stunted transcriptional expression in HM cells

[0130] In both hESC-CM cell lines, transcriptional expression of CYP2J2 was superlatively higher than CYP2C8 and CYP2C9, the other two primary P450 epoxygenases. In WT cells, CYP2I2 demonstrated 29-fold (p < 0.001) and 4938-fold (p < 0.001) higher expression than CYP2C8 and CYP2C9 respectively. Similarly, in HM cells, CYP2J2 exhibited 44-fold (p < 0.001 ) and 1771 -fold (p < 0.001 ) greater expression than CYP2C8 and CYP2C9, respectively. Notably, mRNA expression of CYP2J2 decreased by 32% (p < 0.001) in HM cells compared to WT cells (Figure 3).

[0131] Example 4 - Results: Fluorescent probe substrate ER-BnXPI enabled in-vitro imaging of CYP2.J2 and demonstrated that HM hESC-CMs had reduced Vmax with comparable Kmof ER-BnXPI demethylation

[0132] The fluorescent probe substrate ER-BnXPI was utilized to characterize CYP2J2 activity in hESC-CMs. Given the specific production of fluorescent metabolite ER-OXPI byCYP2J2

[0077] (Figure 4A), the probe displayed potential for both qualitative imaging of CYP2J2 and direct quantitation of CYP2J2 functional activity since the magnitude of fluorescence was indicative of the rate of CYP2J2-mediated ER-OXPI formation. Prior to experimentation, the cytotoxicity of ER-BnXPI in hESC-CMs was evaluated (Figure 4B). Incubation of hESC-CMs with ER-BnXPI generated strong fluorescent emissions that facilitated localization of functional CYP2J2 in hESC-CMs (Figure 4C). Next, by treating hESC-CMs with various concentrations of ER-BnXPI (0.05-25 pM) and evaluating fluorescent emissions of individual cells by flow cytometry, the kinetic parameters of CYP2J2-mediated ER-BnXPI demethylation were characterized (Figure 5). HM hESC-CMs demonstrated a 31% decrease in surrogate Vmax compared to WT cells (p < 0.05), while apparent Kmvalues remained comparable (p = 0.55) between the 2 cell lines.

[0133] Example 5 - Results: HM hESC-CMs exhibited substantially shorter and more variable basal beat-to-beat (BTB) intervals, and were more susceptible to danazol-induced BTB variability

[0134] Capitalizing on the spontaneous contractility of the present hESC-CM genotype models, BTB intervals of hESC-CMs were measured via fluorescent imaging of calcium transients to investigate the cardio-electrophysiological impact of the SNP, both basally and when challenged by a known CYP2J2 inhibitor. Under basal conditions, HM hESC-CMs exhibited rates of contraction over 3-fold faster than that of WT hESC-CMs (p < 0.001), accompanied by 1.6-fold greater variability (relative standard deviation) in BTB intervals (p < 0.001) (Figure 6A). Furthermore, following 24 h incubation with CYP2J2 reversible inhibitor danazol (Figure 14) [6,42,47,50], no statistically significant alterations to mean BTB interval (p = 0.43) or BTB interval variability (p = 0. 18) were observed for WT hESC-CMs. Conversely, mean BTB interval and BTB interval variability increased by 1.2-fold (p < 0.001) and 1 7-fold (p < 0.001) respectively for HM hESC-CMs (Figure 6C).

[0135] Example 6 - Results: The transcriptional expression of CYP2J2 was stunted in both CYP2J2*l / *7 and CYP2J2*7!*7 hESC-CMs

[0136] Consistent with previous findings, CYP2J2 was the predominant P450 epoxygenase in CYP2J2*1 / *1 WT hESC-CMs, with its transcriptional expression 8.2-fold higher than CYP2C8 and 176.2-fold higher than CYP2C9. However, as shown in Figure 15, in CYP2J2*l / *7 (heterozygous) and CYP2J2*7 / *7 (homozygous) hESC-CMs, CYP2I2 mRNA expression was significantly reduced by 84.8% and 85.8%, respectively, compared to WT (p < 0.05). This substantial reduction led to CYP2.I2 no longer being the predominant P450epoxygenase, with CYP2C8 emerging as the major expressed P450 epoxygenase in these genotyped cell lines. Notably, CYP2C9 expression remained low across all groups.

[0137] Example 7 - Results: Functional characterization using ER-BnXPI demonstrated reduced Vmax with comparable Kmin both CYP2J2*l / *7 and CYP2J2*7 / *7 hESC-CMs

[0138] To assess the impact of CYP2J2*7 allele on CYP2J2 catalytic activity, hESC-CMs were treated with varying concentrations of ER-BnXPI (0.05-25 pM) and fluorescence emissions of individual cells were measured via flow cytometry to determine the kinetic parameters of CYP2J2-mediated ER-BnXPI demethylation (Figure 16). Compared to CYP2J2*1 / *1 WT hESC-CMs, both CYP2J2* l / *7 (heterozygous) and CYP2J2*7 / *7 (homozygous) hESC-CMs demonstrated a significant reduction in surrogate Vmai by 27% and 31%, respectively (p < 0.05) (Table 8). However, apparent Kmvalues remained comparable across all groups (p = 0.55), suggesting that the substrate binding affinity was not significantly altered despite the reduction in maximal enzymatic capacity.

[0139] Table 8. Michaelis-Menten kinetic parameters for CYP2J2-mediated demethylation of ER-BnXPI. Apparent Vmax = maximal velocity based on relative fluorescent units of surrogate metabolite, ER-OXPI, Km= Michaelis-Menten constant or substrate concentration at 50% of maximal velocity.

[0140] Example 8 - Results: CYP2J2* l / *7 and CYP2J2*7 / *7 hESC-CMs exhibited lower and more variable basal beat-to-beat (BTB) intervals, with CYP2J2*7 / *7 hESC-CMs being more susceptible to danazol-induced BTB variability

[0141] To investigate the cardio-electrophysiological impact of the CYP2J2 allele, the spontaneous contractility of the present hESC-CM genotype models was capitalized on, and BTB intervals of hESC-CMs were measured via fluorescent imaging of calcium transients, both under basal conditions and following exposure to a known CYP2J2 inhibitor, danazol.

[0142] Under basal conditions, both CYP2J2 *l / *7 and CYP2J2 *7 / *7 hESC-CMs exhibited rates of contraction over 2-fold faster than that of WT hESC-CMs (p < 0.001) (Figure 17A). This was accompanied by a 1.2-fold (CYP2J2*l / *7) and a 1.3-fold (CYP2J2*7 / *7) increase in BTB variability (relative standard deviation) compared to WT hESC-CMs (p < 0.05 and p < 0.001 ) (Figure 17B) (Table 9).

[0143] Table 9. Basal BTB intervals and variability in hESC-CMs. Basal mean BTB interval (seconds) and BTB variability (relative standard deviation) in CYP2J2*1 / *1, CYP2J2*l / *7, and CYP2J2*7 / *7 hESC-CMs before treatment. Values are presented with the corresponding sample size (n).Cell Line Basal Mean BTB Interval Basal Mean BTB Variability2 20 0 11CYP2J2‘1f1 hESC-CMs ^°31 1 )(n"31 1 )

[0144] Following 24-hour incubation with CYP2J2 reversible inhibitor danazol, CYP2J2*7 / *7 hESC-CMs exhibited the greatest drug-induced proarrhythmic risk, as reflected by the highest BTB variability (relative standard deviations of 0.27) compared to CYP2J2*1 / *1 WT hESC-CMs (0.20, p < 0.001). This suggested that CYP2J2*7 / *7 hESC-CMs are more susceptible to drug-induced proarrhythmia. Although an increase in BTB variance was also observed in CYP2J2*l / *7 hESC-CMs both basally (0.13) and post-danazol treatment (0.22), these differences were not statistically significant compared to CYP2J2*1 / *1 WT hESC-CMs (Figure 17C) (Table 10).

[0145] Currently, the present CYP2J2 *1 / *1 WT and CYP2J2 *7 / *7 hESC-CM datasets each contain over 100 data points, whereas the CYP2J2*l / *7 dataset consists of over 50 data points.

[0146] Table 10. Effect of danazol treatment on BTB variability in hESC-CMs. Mean BTB variability (relative standard deviation) in CYP2J2* 17*1, CYP2J2*l / *7, and CYP2J2*7 / *7 hESC-CMs under control conditions (DMSO) and after 24-hour treatment with danazol. Values are presented with the corresponding sample size (n).

[0147] Example 9 - Results: CYP2J2-mediated AA epoxygenation is impaired in CYP2 J 2*7 -bearing hESC-CMs

[0148] Although ER-BnXPI serves as a useful surrogate for assessing CYP2J2 functional activity due to the clear fluorescence read-out, the capacity of CYP2J2 in metabolizing physiologically relevant substrates, particularly AA to EETs, remains unelucidated. Given the critical role of EETs in regulating cardiac electrophysiology, with its elevated levels mitigating arrhythmic risk and reduced synthesis being associated with drug-induced proarrhythmia, the functional impact of CYP2J2*7 on EET biosynthesis was next investigated. To this end, 14,15- EET, the predominant regioisomer produced by CYP2J2, was quantitated in hESC-CMs treated with increasing concentrations of AA (1-100 pM). Kinetic analysis revealed that AA epoxygenation followed classical Michaelis-Menten kinetics across all genotypes (Figure 18). Notably, both CYP2J2*l / *7 and CYP2J2*7 / *7 hESC-CMs exhibited a 70% reduction incompared to CYP2J2*1 / *1 control, while their Kmvalues remained statistically comparable (Table 11), indicating the preserved substrate affinity but impaired catalytic efficiency of AA epoxygenation. These reductions in Vmax ultimately translated into a 45% and 38% decrease in the calculated apparent intrinsic clearance (CL,„f) for CYP2J2*l / *7 and CYP2J2*7 / *7 hESC- CMs, respectively, which provide a potential mechanistic explanation for the heightened proarrhythmic risks observed in CYP2J2*l / *7 and CYP2J2 *77*7 hESC-CMs.

[0149] Table 11. Michaelis-Menten kinetic parameters for CYP2J2-mediated epoxygenation of AA. Vmax = maximal velocity based on amounts of surrogate metabolite, 14,15-EET, Km= Michaelis-Menten constant or substrate concentration at 50% of

[0150] Kinetic Parameters of CYP2J2-Mediated Arachidonic Acid Epoxy genation in hESC- CMs

[0151] Both CYP2J2*1 / *1 and CYP2J2*7 / *7 hESC-CMs were incubated in a 37 °C, 5% CO2 incubator for 24 h with varying concentrations of astemizole (1, 5, 10, 20, 40, 60, 80, 100 pM). 2 rnL aliquots of culture medium were quenched with 2 mL icc-cold EA containing 100 11M of 4-HBP internal standard, 0.005 mg / mL BHT as the anti-oxidant and 5% acetic acid at the end of 24 h. Quenched aliquots of media were centrifuged at 18,000 g, 4 °C for 30 min, and 1500 pL of supernatant was retrieved, dried and reconstituted in cold ACN. Reconstituted samples were analysed by liquid chromatography -tandem mass spectrometry (LC-MS / MS, Table 12). Calibration standards in medium were processed similarly to samples whereby the major epoxide 14,15-EET and 4-HBP were spiked in blank GF AM medium (Figure 19). For analysis, the pcak-arca-ratio of 14,15-EET against internal standard 4-HBP was normalized against protein concentration, adjusted to the rate of formation and quantified based on the calibration curve, and plotted against astemizole concentration to derive its Kmand Vmax. Since the cellular volume of H7 hESC-CMs has not been previously reported, the volume of AC 16, an immortalized human cardiac cell line, was taken as a surrogate to calculate the apparent intrinsic clearance (CLint) < / .). Graphical analysis was performed with GraphPad Prism 8.0.2.

[0152] LC-MS / MS Methods for Detection of 14,15-EET

[0153] All samples were analyzed using the LC-MS / MS system consisting of an Agilent 1290 Infinity ultra-high pressure liquid chromatography (Agilent Technologies Inc., Santa Clara, CA) interfaced with QTRAP® 5500 mass spectrometer (MS / MS) (AB SCIEX,Framingham, MA). Chromatographic separation was achieved with an Atlantis Premier BEH C18 AX, 1.7 pm, 2.1 x 100 mm (Waters, Milford, MA).

[0154] Referencing the LC-MS / MS method developed by Ng et al.

[0109] , the aqueous mobile phase (A) was 7.5 mM ammonium acetate in a mixture of ACN and water (1: 1), whereas the organic mobile phase (B) was 7.5 mM ammonium acetate in a mixture of IPA and ACN (3:2). Mobile phases were delivered at a flow rate of 0.45 mL / min. The column and sample temperatures were set at 60 °C and 4 °C respectively. The gradient elution conditions were as follows: linear gradient from 50% to 95% B (0 - 4.00 min), linear gradient from 95% to 50% B (4.00 - 4.10 min), isocratic at 50% B (4.10 - 5.50 min). All analytes were detected in negative electrospray ionization (ESI) mode. The source-dependent MS parameters are as follows: ion spray voltage = 5500 V; source temperature = 500°C; curtain gas (CUR) = 25 psi; ion source gas 1 (sheath gas) = 30 psi; ion source gas 2 (drying gas) = 30 psi. The MRM transitions and compound-dependent MS parameters of the analytes are summarized in Table 12.

[0155] Table 12. Compound-dependent MS parameters of 14,15-EET and 4-HBPQI : mass of parent ion; Q3: mass of daughter ion; DP: declustering potential; EP: entrance potential; CE: collision energy; CXP: cell exit potential.

[0156] Chromatographic peak integration was performed using SCIEX OS version 1.5.0.23389 (AB SCIEX, Framingham, MA). For all LC-MS / MS analyses, the peak area of the analyte was expressed as a ratio to the peak area of the internal standard.

[0157] Data Analysis

[0158] All data analysis was conducted using GraphPad Prism software (version 9.5.1, GraphPad Software, Inc., San Diego, CA). Specifically, apparent Kmand Vmaxwere determined through nonlinear regression analysis of the kinetic data by applying the Michaelis -Menten model.

[0159] Example 10 - Results: CYP2J2*l / *7 predisposes heart failure (HF) patients to more non-atrial fibrillation electrocardiographic (non-AF ECG) abnormalities

[0160] After establishing that the CYP2I2*7 allele alters cardiac electrophysiology in vitro, whether this SNP is clinically associated with ECG abnormalities in patients was next investigated. To this end, clinical validation was subsequently performed by combining two large Asian heart failure cohorts, namely the SHOP cohort

[0106] and the ATTRaCT cohort

[0107] , with case-matched healthy controls. Consistent with SG10K population data (5.4%), CYP2J2*7 allele frequency was calculated to be 5.26%, with most carriers harbouring the CYP2J2*l / *7 genotype. ECG abnormalities were significantly enriched among HF patients (Fisher’s exact test p < 0.01) (Figure 20a). While overall ECG abnormality rates did not differ significantly between CYP2J2*1 / *1 and CYP2J2*l / *7 patients, non-atrial fibrillation (non- AF) ECG abnormalities were significantly enriched in CYP2J2*l / *7 HF patients (Fisher’s exact test p < 0.01) (Figure 20b). Stratification by New York Heart Association (NYHA) class further revealed the most pronounced electrophysiological divergence in class III patients, where CYP2J2 *l / *7 patients exhibited AV block and previous pacemaker insertion but not AF, in contrast to a 21.8% AF prevalence among CYP2J2*1 / *1 patients (Fisher’s exact test p = 0.02) (Figure 20c). Binomial logistic regression further identified CYP2J2*l / *7 genotype (p < 0.01), along with Malay ethnicity (p = 0.02), heart rate (p < 0.01), and prior stroke (p = 0.04), as independent predictors of non-AF ECG phenotypes in HF (Table 13).[00161 J Table 13. Binomial regression of factors influencing abnormal ECG presentation - AF vs non-AF presentations

[0162] Study population

[0163] The study cohort was derived from the Singapore Heart Failure Outcomes and Phenotypes (SHOP)

[0106] cohort and the Asian Network for Translational Research and Cardiovascular Trials (ATTRaCT) cohort

[0107] study, with the source dataset including both electronic health records (EHR) and genomic data stored in the BioMed Data Access Repository (BioMedDAR). All participants provided informed consent, and the study protocol was approved by the institutional ethics review boards [CIRB 2015-2194, CIRB 2015-2787, CIRB 2019-2046, DSRB 2013 / 00105], A total of 1,386 individuals with both EHR and genomic data were included in the final analysis.

[0164] Diplotype calling for CYP2J2 polymorphisms

[0165] Valiant call format (VCF) files, previously curated using the GcnomcAnalysisToolKit (GATK, v4.0.6.0) best practices workflow, were obtained from BioMedDAR in alignment with the Research Collaboration Agreement (RCA). The VCF files were subsequently truncated to retain only variants on chromosome 1, where the CYP2J2 gene is located. Diplotype calling was performed using ALDY 4.0 to assign CYP2.T2 genotypes for further analysis.

[0166] Statistical analysis

[0167] All statistical analysis was performed using R version 4.1.0 with the tidyverse and ggpubr packages. Group comparisons of categorical variables were performed using Fisher’s exact test. To correct for multiple hypothesis testing, Bonferroni correction was applied for p- value adjustments.

[0168] Example 11 - Discussion

[0169] As the predominant P450 expressed in cardiomyocytes, the pivotal role of CYP2J2 in regulating cardio-electrophysiology, principally through its biotransformation of AA to cardioprotective EETs, remains irrefutable. Given the high allele frequency of the CYP2J2*7 SNP and its association with various adverse cardiovascular events, this investigation focusedon characterizing the direct cellular impact of the SNP on CYP2J2 transcriptional expression and enzymatic activity, and by extension the effect on intrinsic cardiomyocyte contraction control and CYP2J2 inhibitor-induced arrhythmia.

[0170] Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have gained tremendous popularity as alternative in vitro disease models. Possessing unlimited replicative abilities inherent to immortalized cell lines, combined with the expression profile and spontaneous contractility of primary cardiomyocytes, without the cost and extrapolative uncertainty associated with animal models, hiPSC-CMs are an adaptable platform uniquely capable of emulating the native cardiac milieu necessary for reproduction of cellular disease states associated with various mutations, rendering them an invaluable tool for elucidating fundamental molecular mechanisms underpinning hereditary cardiomyopathies [78-84], Hence, leveraging on their ability to recapitulate relevant electrophysiological phenotypes, hESC-CM genotype models harboring the CYP2J2*7 SNP were developed, and further engineered to constitutively express the Ca2+channel reporter GCaMP6s to streamline characterization of hESC-CM electrophysiology for proarrhythmic assessment.

[0171] To achieve directed differentiation of hESCs into cardiomyocytes, the GiWi protocol

[0074] , which utilized small molecules to chronologically modulate canonical Wnt signalling, was referenced. This 2-step methodology involved initial indirect Wnt activation through impediment of glycogen synthase kinase 3 (GSK-3[3)-mediated Wnt degradation to promote mesodermal differentiation, and subsequent direct Wnt inhibition leading to cardiac lineage commitment [75,85-87], However, this technique was inconsistent and regularly resulted in massive cell death during early differentiation (D1-D5), a sentiment echoed by numerous groups [75,88,89], Thus, taking inspiration from Meng et al.

[0075] , a low maintenance dose of GSK-3P inhibitor was incorporated to prolong Wnt activation between D 1 -3, and supplemented hESCs with insulin on DO to improve cell viability. Insulin supplementation was later ceased as insulin suppressed mesodermal commitment [90,91], and the introduction of the maintenance dose, combined with initiation of differentiation only at higher hESC confluency, was already adequate in substantially elevating cell survival. Notably, a maturation phase was also assimilated where RPMI B+ was substituted for glucose-free, fatty acid-enriched culture media (GFAM) after DI 9. By directing metabolic diversion towards fatty acid |3-oxidation typical of mature primary cardiomyocytes, concurrent metabolic maturation and cardiomyocyte selection could be achieved

[0076] , This phase was instrumental, as prior methods to purify hESC-CMs, including fluorescent-assisted and magnetic-assisted cell sorting, wereunable to consistently yield hESC-CMs suitable for downstream assays, with few purified batches surviving post-purification and none retaining spontaneous contractility. This could be due to the inability of hESC-CMs to re-establish gap junctions and consequently ion flow between individual cardiomyocytes upon initial disruption during cellular dissociation. To account for the perpetually evolving metabolic and electrophysiological profile with continued maturation [92-95], characterization experiments were restricted to hESC-CMs between D25- 30 of differentiation. Ultimately, these optimizations enabled the consistent production of relatively pure and spontaneously contracting hESC-CMs.

[0172] Regarding CYP2J2 expression, the transcriptional analyses confirmed CYP2J2 as the predominant P450 epoxygenase in both WT and HM hESC-CMs, congruent with prior studies performed by Michaud et al. [5] and Evangelista et al. [6] on primary human cardiomyocytes. Furthermore, a direct reduction in CYP2J2 mRNA transcript levels of approximately 30% in HM cells compared to WT controls was demonstrated. This concurred with the inaugural investigation by Spiecker et al., which indirectly assessed the SNP’s transcriptional impact by conjugating the CYP2J2*7 mutant promoter to a luciferase reporter, ultimately reporting a 48% reduction in promoter activity

[0062] ,

[0173] Subsequently, since additional factors including translational regulation and protein degradation could perturb the mRNA-protein correlation [96-99], kinetics experiments to validate the SNP’s translational impact on enzymatic activity were performed. Given SNP’s localization in the proximal promoter, it was anticipated that HM hESC-CMs would display diminished maximal rates of enzymatic metabolism (Vmax) due to stunted expression, while maintaining unaltered Kmindicative of unchanged enzymatic structure, function and affinity towards its respective substrates.

[0174] To evaluate this postulation, ER-BnXPI was employed as a probe substrate in hESC- CMs with fluorescent emissions of CYP2J2-specific metabolite ER-OXPI serving as a surrogate for CYP2J2 functional activity to enable characterization of kinetic parameters. These experiments revealed a decrease in surrogate Vmax of ER-BnXPI demethylation in HM hESC- CMs relative to WT controls while apparent Kmremained unchanged, thus supporting the hypothesis. Notably, as fluorescent emissions of individual hESC-CMs could be directly monitored via flow cytometry to gauge CYP2J2 activity post-incubation, the application of ER- BnXPI enabled truncation of the sophisticated biological sample preparation required for classical recombinant enzyme or cellular metabolic assays, including cell lysis, protein precipitation, metabolite extraction and enrichment, and quantification. Taken together, ER-BnXPI drastically simplified the characterization of CYP2J2 functional activity in hESC-CMs and abated the considerable experimental variability associated with these complex procedures. However, ER-BnXPI notably demonstrated markedly elevated cytotoxicity in the hESC-CMs (Figure 4B) compared to the human cancer cell lines employed by the original developers

[0077] , resulting in the reported apparent Kmvalues (WT = 3.82 pM, HM = 3.17 pM) exceeding the ICso for both cell lines (WT = 0.94 pM, HM = 0.30 pM). Such divergent cytotoxic potencies could have arose due to differential expression of efflux transporters or occurrence of escape survival pathways between cell lines, given the tendency for cancer cells to develop resistance mechanisms [100-102], To compensate for these viability concerns, quantitation of single-cell fluorescent emissions was limited to live single cells via flow cytometric gating, under the reasonable assumption that CYP2J2 activity in surviving cells would be unaffected by the probe’s cytotoxicity. In addition, arachidonic acid (AA), the endogenous substrate, was used as the substrate to measure and compare the activities of CYP2J2 WT and its *7 variants. Results showed that CYP2J2-mediated AA epoxygenation is impaired in CYP2J2*7-bearing hESC- CMs.

[0175] Next, the present study established that the CYP2J2*7 SNP not only significantly elevated the basal contraction rate and variability of hESC-CMs, but also amplified susceptibility to CYP2J2 inhibitor-induced arrhythmia as only HM hESC-CMs exhibited statistically significant alterations in mean BTB interval and BTB interval variability upon exposure to CYP2J2 inhibitor danazol. These findings imply that human carriers of CYP2J2*7 may be inherently predisposed to cardiac arrhythmia, and also harbor an elevated proarrhythmic risk when further exposed to CYP2J2-inhibiting xenobiotics. While these observations were aligned with the initial hypothesis, the momentous extent of the perturbation in basal contractility was unprecedented, especially considering the current lack of reported clinical association between CYP2J2*7 and arrhythmia. Hence, caution is warranted in labelling CYP2J2*7 as a tachycardia genotype, particularly given the limited clinical translatability of the hESC-CM model arising from its immaturity relative to adult primary cardiomyocytes. Despite tremendous advancements in recent years, modern hiPSC-CMs still bear greater resemblance to fetal cardiomyocytes, especially with regard to morphology, metabolism and electrophysiology [92-95,103], This is epitomized by the WT hESC-CM controls exhibiting a contraction rate of 23 beats-per-minute (bpm) versus the normal human heart rate ranging between 60-100 bpm [104,105], emphasizing the electrophysiological disparity between the in vitro model and in vivo cardiovascular environment.

[0176] In addition, both CYP2J2*l / *7 and CYP2J2*7 / *7 hESC-CMs exhibited reduced transcriptional expression, catalytic activities and heighted intrinsic and drug-induced proarrhythmic risks compared to CYP2J2 WT hESC-CMs (Figure 15-17). These findings strongly implicate CYP2J2*7 as a predisposing factor to proarrhythmia and CYP2J2-inhibitor induced proarrhythmia. CYP2J2*l / *7 was also shown to predispose heart failure (HF) patients to more non-atrial fibrillation electrocardiographic (non-AF ECG) abnormalities according to the clinical data disclosed herein.

[0177] In conclusion, these findings represent the inaugural illumination of the electrophysiological impact of the CYP2J2*7 SNP, and paves the framework for advancing mechanistic comprehension into the role of CYP2J2 in both intrinsic cardiomyocyte contractility control and drug-induced proarrhythmia (Figure 7). Critically, this study furnishes vital insights for re-evaluating the clinical risk of CYP2J2 reversible inhibitor or irreversible inactivator-induced proarrhythmia, particularly in CYP2J2*7-carrying patients who are innately more vulnerable.

[0178] Industrial Applicability

[0179] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

[0180] The industrial application of genotyping CYP2J2*7 is applicable to the (1) diagnosis of patients who have a proarrhythmic substrate and potentially susceptible to drug-induced arrhythmic risk (precision diagnosis), (2) optimization of pharmacotherapy of regulatory approved drugs that arc CYP2J2 inhibitors or inactivators where CYP2J2*7 carriers arc contraindicated (precision medicine) and (3) clinical studies that implicate CYP2J2*7 as an efficacy or safety biomarkers.References[1] Guengerich FP. Mechanisms of Cytochrome P450-Catalyzed Oxidations. ACS Catal 2018;8:10964-76. https: / / doi.org / 10.1021 / acscatal.8b03401.[2] Anne M McDonnell CHD. Basic Review of the Cytochrome P450 System 2013;4:263- 8. https: / / doi.Org / 10.6004 / jadpro.2013.4.4.7.[3] Zhao M, Ma J, Li M, Zhang Y, Jiang B, Zhao X, et al. Cytochrome P450 Enzymes and Drug Metabolism in Humans. IJMS 2021,22: 12808. https: / / doi.org / 10.3390 / ijms222312808.[4] Berlin DS, Sangkuhl K, Klein TE, Altman RB. PharmGKB summary: cytochrome P450, family 2, subfamily J, polypeptide 2 CYP2J2. Pharmacogenetics and Genomics 2011;21 :308— 11. https: / / doi.org / 10.1097 / FPC.0b013e32833dl011.[5] Michaud V, Frappier M, Dumas M-C, Turgeon J. Metabolic Activity and mRNA Levels of Human Cardiac CYP450s Involved in Drug Metabolism. PLoS ONE 2010;5:el5666. https: / / doi.org / 10.1371 / journal.pone.0015666.[6] Evangelista EA, Kaspera R, Mokadam NA, Jones JP, Totah RA Activity, Inhibition, and Induction of Cytochrome P450 2J2 in Adult Human Primary Cardiomyocytes. Drug Metab Dispos 2013 ;41 :2087-94. https: / / doi.org / ! 0.1124 / dmd.113.053389.[7] Evangelista EA, Aliwarga T, Sotoodehnia N, Jensen PN, McKnight B, Lemaitre RN, et al. CYP2J2 Modulates Diverse Transcriptional Programs in Adult Human Cardiomyocytes. Sci Rep 2020;10:5329. https: / / doi.org / 10.1038 / s41598-020-62174-w.[8] DeLozier TC, Kissling GE, Coulter SJ, Dai D, Foley JF, Bradbury JA, et al. Detection of Human CYP2C8, CYP2C9, and CYP2J2 in Cardiovascular Tissues. Drug Metab Dispos 2007;35:682-8. https: / / doi.org / 10.1124 / dmd.106.012823.[9] Wang H, Jiang Y, Liu Y, Lin C, Cheng G, Chen X, et al. CYP2J2*7 single nucleotide polymorphism in a Chinese population. Clinica Chimica Acta 2006;365: 125-8. https: / / doi.Org / 10.1016 / j.cca.2005.08.007.

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Claims

1. Claims1. A method of identifying a subject who has or is predisposed to cardiac arrhythmia, the method comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein the presence of the allele indicates that the subject has or is predisposed to cardiac arrhythmia.

2. The method of claim 1, wherein the cardiac arrhythmia is intrinsic arrhythmia or drug- induced proarrhythmia.

3. The method of claim 2, wherein the drug is a CYP2J2 reversible inhibitor or irreversible inactivator.

4. The method of claim 3, wherein the CYP2J2 reversible inhibitor or irreversible inactivator is selected from the group comprising danazol, dronedarone, amiodarone, bepridil, isradipine, nicardipine, verapamil, lovastatin, simvastatin, ranolazine, quinapril, telmisartan, erlotinib, nilotinib, imatinob, gefitinib, apatinib, motesanib, vatalanib, sorafenib, vanderanib, ibrutinib, osimertinib, infigratinib, cisapride, astemizole, loratadine, terfenadine, ondansetron, domperidone, droperidol, clozapine, haloperidol, pimozide, risperidone, thioridazine, omeprazole, lansoprazole, orphenadrine, clotrimazole, miconazole, ketoconazole, clomiphene, tamoxifen, amodiaquine, mefloquine, ivermectin, albendazole, quercetin, fluoxetine, fluvoxamine, paroxetine methadone, cyclobenzaprine, nortriptyline, budesonide, ritonavir, flunarizinc, vandetanib, piperine, mevinolin, LKY-047, and derivatives thereof.

5. The method of claim 1, wherein the sample is blood, saliva, or buccal cells.

6. The method of claim 5, wherein the nucleic acid is a nucleic acid extract from the sample.

7. The method of claim 6, further comprising preparing the nucleic acid extract from the sample prior to the testing.

8. The method of claim 1, wherein the testing comprises nucleic acid amplification.

9. The method of claim 8, wherein the nucleic acid amplification is carried out by polymerase chain reaction.

10. The method of claim 1, wherein the testing is performed using restriction fragment length polymorphism (RFLP), sequencing, 5' nuclease digestion, molecular beaconassay, oligonucleotide ligation assay, size analysis, single- stranded conformation polymorphism analysis, or denaturing gradient gel electrophoresis (DGGE).

11. The method of claim 1, wherein said testing is performed using an allele-specific method selected from the group consisting of allele -specific probe hybridization, allelespecific primer extension, or allele-specific amplification.

12. The method of claim 1, wherein subject is human.

13. The method of claim 12, wherein the human is homozygous for the allele.

14. The method of claim 12, wherein the human is heterozygous for the allele.

15. The method of claim 12, wherein the human is wildtypc CYP2J2*1 / *1, and docs not have the allele.

16. The method of claim 1, wherein the subject did not have cardiac arrhythmia prior to the testing.

17. The method of claim 1, wherein the subject did have cardiac arrhythmia prior to the testing.

18. The method of claim 1, further comprising administering a CYP2J2 reversible inhibitor or irreversible inactivator to the subject who does not have the allele.

19. The method of claim 1, further comprising reducing the dosage or optimizing the dosing regimen of a CYP2J2 reversible inhibitor or irreversible inactivator or administering a therapeutically efficacious dose of a therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator to the subject who has the allele.

20. The method of claim 18 or 19, wherein the CYP2J2 reversible inhibitor or irreversible inactivator is selected from the group comprising danazol, dronedarone, amiodarone, bepridil, isradipine, nicardipine, verapamil, lovastatin, simvastatin, ranolazine, quinapril, telmisartan, erlotinib, nilotinib, imatinob, gefitinib, apatinib, motesanib, vatalanib, sorafenib, vanderanib, ibrutinib, osimertinib, infigratinib, cisapride, astemizole, loratadine, terfenadine, ondansetron, domperidone, droperidol, clozapine, haloperidol, pimozide, risperidone, thioridazine, omeprazole, lansoprazole, orphenadrine, clotrimazole, miconazole, ketoconazole, clomiphene, tamoxifen, amodiaquine, mefloquine, ivermectin, albendazole, quercetin, fluoxetine, fluvoxamine, paroxetine methadone, cyclobenzaprine, nortriptyline, budesonide, ritonavir, flunarizine, vandetanib, piperine, mevinolin, LKY-047, and derivatives thereof.

21. A method of treating or preventing cardiac arrhythmia in a subject having or predisposed to cardiac arrhythmia, comprising: a. obtaining a sample from the subject; b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein when the allele is present, the method further comprising administering a reduced dose or optimizing the dosing regimen of a CYP2J2 reversible inhibitor or irreversible inactivator compared to when the allele is absent, or administering a therapeutically efficacious dose of a therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator to the subject.

22. A method for establishing a prognosis for a patient having or predisposed to cardiac arrhythmia and treated by a CYP2J2 reversible inhibitor or irreversible inactivator, comprising: a. obtaining a sample from the subject; and b. testing nucleic acid from the sample for the presence or absence of an allele at a single nucleotide polymorphism (SNP) which is CYP2J2*7; wherein the presence of the allele indicates poor prognosis.

23. The method of claim 22, further comprising administering a reduced dose or optimizing the dosing regimen of a CYP2J2 reversible inhibitor or irreversible inactivator to the subject who has the allele compared to when the allele is absent, or administering a therapeutically efficacious dose of a therapeutic agent from the same therapeutic class that is not a CYP2J2 reversible inhibitor or irreversible inactivator to the subject who has the allele.

24. The method of claim 21 or 22, wherein the cardiac arrhythmia is intrinsic arrhythmia or drug-induced proarrhythmia.

25. The method of claim 24, wherein the drug is a CYP2J2 reversible inhibitor or irreversible inactivator.

26. The method of claim 25, wherein the CYP2J2 reversible inhibitor or irreversible inactivator is selected from the group comprising danazol, dronedarone. amiodarone, bepridil, isradipine, nicardipine, verapamil, lovastatin, simvastatin, ranolazine, quinapril, telmisartan, erlotinib, nilotinib, imatinob, gefitinib, apatinib, motesanib, vatalanib, sorafenib, vanderanib, ibrutinib, osimertinib, infigratinib, cisapride, astemizole, loratadine, terfenadine, ondansetron, domperidone, droperidol, clozapine,haloperidol, pimozide, risperidone, thioridazine, omeprazole, lansoprazole, orphenadrine, clotrimazole, miconazole, ketoconazole, clomiphene, tamoxifen, amodiaquine, mefloquine, ivermectin, albendazole, quercetin, fluoxetine, fluvoxamine, paroxetine methadone, cyclobenzaprine, nortriptyline, budesonide, ritonavir, flunarizine, vandetanib, piperine, mevinolin, LKY-047, and derivatives thereof.

27. The method of claim 21 or 22, wherein the sample is blood, saliva, or buccal cells.

28. The method of claim 27, wherein the nucleic acid is a nucleic acid extract from the sample.

29. The method of claim 28, further comprising preparing the nucleic acid extract from the sample prior to the testing.

30. The method of claim 21 or 22, wherein the testing comprises nucleic acid amplification.

31. The method of claim 30, wherein the nucleic acid amplification is carried out by polymerase chain reaction.

32. The method of claim 21 or 22, wherein the testing is performed using restriction fragment length polymorphism (RFLP) sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, or denaturing gradient gel electrophoresis (DGGE).

33. The method of claim 21 or 22, wherein said testing is performed using an allele-specific method selected from the group consisting of allele -specific probe hybridization, allelespecific primer extension, or allele-specific amplification.

34. The method of claim 21 or 22, wherein subject is human.

35. The method of claim 34, wherein the human is homozygous for the allele.

36. The method of claim 34, wherein the human is heterozygous for the allele.

37. The method of claim 34, wherein the human is wildtype CYP2J2*1 / *1, and does not have the allele.

38. The method of claim 21 or 22, wherein the subject did not have cardiac arrhythmia prior to the testing.

39. The method of claim 21 or 22, wherein the subject did have cardiac arrhythmia prior to the testing.

40. A detection reagent for carrying out the method of claim 1, 21 or 22, wherein said detection reagent is an allele- specific probe or an allele- specific primer.

41. A test kit comprising one or more containers containing the detection reagent of claim 40 and one or more components selected from the group consisting of an enzyme, polymerase enzyme, ligase enzyme, buffer, amplification primer pair, dNTPs, ddNTPs, positive control nucleic acid, negative control, and nucleic acid extraction reagent.

Images