WO2020220136A1

WO2020220136A1 - Detection of biomarkers associated with brugada syndrome

Info

Publication number: WO2020220136A1
Application number: PCT/CA2020/050578
Authority: WO
Inventors: Robert Hamilton
Original assignee: The Hospital For Sick Children
Priority date: 2019-05-02
Filing date: 2020-05-01
Publication date: 2020-11-05
Also published as: JP7547470B2; KR20220034725A; CA3138808A1; JP2022531508A

Abstract

A method of detecting target autoantibodies to actin, connexin-43 and keratin in a mammal is provided. The comprises contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin or with antigenic fragments thereof; and detecting the presence of the target autoantibodies in the sample by detecting binding of the target autoantibodies to each of the antigens. The method is useful to diagnose Brugada Syndrome.

Description

DETECTION OF BIOMARKERS ASSOCIATED WITH BRUGADA

SYNDROME

Field of the Invention

[0001] The present invention generally relates to Brugada Syndrome, and in particular relates to methods of diagnosing Brugada Syndrome.

Background of the Invention

[0002] Brugada Syndrome (BrS) is a heritable autosomal dominant disorder with abnormal electrical activity of the heart. BrS and a related condition called ‘early repolarization syndrome (ERS)’ together are called‘J-wave syndromes’. In Southeast Asia, it is referred to as‘Sudden Unexpected Nocturnal Death Syndrome’, and locally termed‘Lai Tai’ (dreaming, screaming death) in Thailand,‘Tsob Tsuang’ (nightmare death syndrome) in Vietnam,‘Bangungut’ (to rise and moan in sleep) in the Phillipines, ‘Phi Am’ (widow ghost) in Taiwan,‘Pokkuri’ (‘pop off) in Japan,‘Dolyeonsa’ in Korea, ‘Dream Disease’ in Hawaii and‘Sudden manhood death syndrome’ in mainland China.

[0003] BrS is characterized by a coved-type ST-segment elevation in the right precordial leads of the electrocardiogram (ECG), although the appearance of this feature is often transient. The Brugada ECG pattern is typically seen in anterior chest leads over the right ventricular outflow tract (RVOT), either in standard leads V 1 through V3, or modified lead positions placed higher on the chest. In keeping with this localized abnormality, some studies report enlargement as well as inflammation in this area. Also, in keeping with a localized abnormality, atypical electrocardiograms are found on the epicardial surface of the RVOT, and epicardial ablation of this area is able to prevent the ventricular fibrillation episodes associated with Brugada syndrome and normalize the ECG in most patients. BrS is associated with a relatively high risk of sudden death in young adults (particularly males), and occasionally in children and infants. Events are exacerbated by fever, alcohol, large meals, high vagal tone, and certain medications.

[0004] The prevalence of BrS is estimated to be 1 :2000 persons, or more than about

18,000 persons in Canada. It is much more prevalent in people of Southeast Asia. Studies have shown a prevalence of 0.05 - 0.6% of ECGs compatible with Brugada syndrome in regions such as Japan. Brugada syndrome is responsible for 4%-12% of unexpected sudden deaths and up to 20% of all sudden death in individuals with an apparently normal heart. In Southeast Asia, it is the second highest cause (following accidents) of death of men under age 40 years.

[0005] There has been no simple, accurate test for BrS, resulting in a failure to diagnose, evaluate or manage the majority of the estimated 18,000 affected individuals in Canada. Clinical identification is dependent on a specific electrocardiographic pattern which is often transient or requires drug provocation. Genetic diagnosis fails to identify 75% of affected patients/families. Mutations of the SCN5A sodium channel have been identified in 25% of patients, but no other genetic causes have acceptable evidence for causality. Identifying individuals at risk for developing disease is difficult. Lack of a simple, accurate test that can identify pre-clinical disease makes cascade screening (a major tool in other inherited arrhythmia conditions) difficult in BrS. Although major symptoms identify a small group of high-risk individuals, and a spontaneous type 1 Brugada ECG pattern identifies a mild risk, risk stratification in BrS patients is incomplete.

[0006] Thus, it would be desirable to develop a simple and sensitive test sufficient to diagnose BrS.

Summary of the Invention

[0007] A unique autoantibody signature against specific proteins has now been identified which is associated with Brugada syndrome, and the determination thereof is, thus, useful for the diagnosis and, optionally, the treatment of BrS in a mammal.

[0008] Accordingly, in one aspect of the invention, a method of detecting target autoantibodies to actin, connexin-43 and keratin in a mammal is provided comprising:

1) contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin antigens, or with antigenic fragments thereof, to bind with the target autoantibodies; and

2) detecting the presence of the target autoantibodies in the sample by detecting binding of the target autoantibodies to each of the antigens. [0009] In another aspect of the invention, a method of diagnosing Brugada syndrome in a mammal is provided. The method comprises the steps of:

1) contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin antigens, or with antigenic fragments thereof to bind

autoantibodies to each of the antigens;

2) detecting the presence of autoantibodies to each of actin, connexin-43 and keratin by detecting binding of the autoantibodies to each of the actin, connexin-43 and keratin antigens; and

3) diagnosing the mammal with Brugada syndrome when the presence of the target autoantibodies is detected.

[0010] In a further aspect of the invention, a method of diagnosing and treating

Brugada syndrome in a mammal is provided. The method comprises the steps of:

1) contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin antigens or with antigenic fragments thereof;

2) detecting the presence of autoantibodies to each of actin, connexin-43 and keratin by detecting binding of the autoantibodies to each of the actin, connexin-43 and keratin antigens;

3) diagnosing the mammal with Brugada syndrome when the presence of each of the target autoantibodies is detected; and

4) treating the mammal with one or more of an implantable cardioverter defibrillator, catheter ablation and medication selected from Class IA antiarrhythmics, b- Adrenergic agonists and phosphodiesterase III inhibitors.

[0011] These and other aspects of the invention will become apparent by reference to the following detailed description.

Brief Description of the Figures

[0012] Figure 1 illustrates the amino acid sequence of each of human alpha 1 cardiac muscle actin and human alpha 1 skeletal muscle actin; [0013] Figure 2 illustrates the amino acid sequence of keratin 24;

[0014] Figure 3 illustrates the amino acid sequence of connexin-43;

[0015] Figure 4 is a schematic illustrating a method of identifying Brugada autoantibodies;

[0016] Figure 5 illustrates the results of a Western Blot analysis on a Brugada syndrome sample and a healthy control sample; and

[0017] Figure 6 graphically illustrates the results of Enzyme-Linked Immuno-

Sorbent Assays (ELISA) demonstrating antibodies to (A) connexin-43, (B) cardiac a-actin, (C) skeletal a-actin and (D) keratin-24 in discovery and validation cohorts versus controls.

Detailed Description of the Invention

[0018] In a first aspect, a method of detecting target autoantibodies to actin, connexin-43 and keratin in a mammal is provided. The method comprises contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin antigens or with antigenic fragments thereof, and detecting the presence of the target autoantibodies in the sample by detecting binding of the target autoantibodies to their corresponding antigen.

[0019] The biological sample obtained from the mammal will generally be a serological sample, including whole blood, plasma and serum, but may also be saliva, urine, cerebrospinal fluid and other bodily fluids. Preferred biological samples are those fluids which may be obtained non-invasively. The biological sample may be obtained using methods well-established in the art, and may be obtained directly from the mammal, or may be obtained from a sample previously acquired from the mammal which has been appropriately stored for future use (e.g. stored at 4 °C). An amount of sample of at least about 100 mΐ, e.g. 100 mΐ of diluted human serum (1 : 100 dilution in blocking buffer) may be used to conduct the present method.

[0020] The term“mammal” is used herein to refer to both human and non-human mammals including, but not limited to, cats, dogs, horses, cattle, goats, sheep, pigs, rodents, and the like. [0021] The sample is contacted with each of actin, connexin-43 and keratin, including the full-length versions of these proteins or antigenic fragments thereof.

[0022] Actin is used herein to refer to mammalian actin, and in particular, the isoforms, alpha-cardiac muscle actin (ACTC1) and/or alpha-skeletal muscle actin (ACTA1). Actin is a highly conserved enzyme that hydrolyzes ATP. The amino acid sequences of human alpha 1 cardiac muscle actin and human alpha 1 skeletal muscle actin are shown in Fig. 1 A) and B), respectively. Functionally equivalent isoforms and variants including amino acid replacements that do not impact function are also encompassed, such as variant proteins from different species. The term“functionally equivalent” refers to isoforms and variants that retain the function of the wild-type protein, not necessarily to the same extent as the wild-type protein, but at least partially, e.g. retains at least about 25% or greater of the activity of the wild-type protein. Antigenic regions of actin, e.g. regions comprising an antibody binding site, may be used to prepare antigenic fragments of actin for use in the present method. In one embodiment, antigenic fragments may be prepared from the N-terminal region of actin, e.g. within 30 amino acids of the N-terminal region. As used herein, the term“antigenic fragment” refers to a fragment of an antigen comprising an epitope to which a target antibody will bind. Antigenic fragments may be various sizes, e.g. from 12-20 amino acids, or greater in size, such as 30, 40, 50 or more amino acids.

[0023] Connexin-43, also referred to as Gap junction alpha-1 protein (GJA1), is a component of gap junctions, intercellular channels that connect adjacent cells to permit the exchange of low molecular weight molecules, such as small ions and secondary messengers to maintain homeostasis. The protein includes N- and C- terminal regions, and multiple transmembrane domains. As used herein, connexin-43 encompasses the mammalian protein. The amino acid sequence of human connexin-43 is shown in Fig. 3. Functionally equivalent isoforms and variants including amino acid replacements that do not impact function are also encompassed such as variant proteins from different species. Antigenic regions of connexin-43 include, for example, the first 230 amino acids of the C-terminal end of the protein.

[0024] Keratin is a fibrous structural protein. As used herein, keratin refers to mammalian keratin, which may include keratins 1 to 20, as well as other keratin compounds such as keratins 23-28, keratins 31-40, etc. The target keratin may be a cardiac- expressed keratin, including type 1 (acidic) keratins such as but not limited to keratin 8, 18, 23 and/or 24. The amino acid sequence of human keratin 24 is shown in Fig. 2. Functionally equivalent isoforms and variants including amino acid replacements that do not impact function are also encompassed such as variant keratin proteins from different species. Antigenic regions of keratin, e.g. regions comprising an antibody binding site, may be used to prepare antigenic fragments of keratin for use in the present method. For example, the antigenic region may comprise amino acids from position 120-400 of keratin, in whole or in part.

[0025] The actin, connexin-43 and keratin antigens, or antigenic fragments thereof, are preferably bound to or immobilized on a solid support, such as a nitrocellulose, polyvinylidene difluoride (PVDF), or cationic nylon membranes to capture the target autoantibodies. As known to those of skill in the art, in order to prevent nonspecific binding on the solid support, free binding sites on the support are blocked using a suitable blocking buffer, e.g. milk, normal serum or purified proteins. The assay employs conditions suitable for autoantibody binding to the proteins, for example, incubation at an appropriate temperature in a suitable buffer. Following a period of time sufficient for binding to occur, the solid support is washed to remove unbound and/or non-specifically bound material. A physiological buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS) may be used, optionally including additives such as a detergent (e.g. 0.05% Tween™ 20).

[0026] The presence of target autoantibodies in the sample is then determined by detecting autoantibody bound to the target actin, connexin-43 and keratin proteins, or bound to antigenic fragments thereof. Methods such as Western Blotting or immunoassays such as Enzyme-Linked Immunoassay (ELISA) may be used for this purpose. Other methods of antibody detection may also be used.

[0027] In one embodiment, bound target autoantibody is detected using a secondary antibody that will bind the target autoantibody and which is detectable. If the sample is a human sample, then an anti-human secondary antibody may be used, for example, anti-human antibody derived from a non-human mammal (e.g. goat, rabbit, mouse, rat, chicken, pig, cow, sheep, donkey) against human immunoglobulin such as immunoglobulin G (IgG). If the sample is a non-human sample, then suitable secondary antibodies, which may be obtained from a different non-human mammal, are used to detect the target autoantibodies. To detect bound target autoantibodies, the sample is contacted with the secondary antibodies under conditions suitable for binding and then washed to remove unbound reagent. The secondary antibody is labelled with any suitable detectable label, either prior or subsequent to autoantibody binding using established protocols. Suitable labels include, but are not limited to, an enzyme label such as glucose oxidase, horseradish peroxidase (HRP) or alkaline phosphatase (AP); a fluorescent label such as ethidium bromide, fluorescein, rhodamine, phycoerythrin, cyanine, coumarin, green fluorescent protein and derivatives thereof; an affinity label such as biotin/streptavidin labelling; or radioactive labels. The presence of the target autoantibodies is then detected by detecting the presence of the selected label using methods known to those of skill in the art. For example, to detect enzyme labels, the appropriate enzyme substrate is added to the sample and enzyme activity is detected by chromogenic, chemiluminescent or fluorescent outputs. Examples of commonly used substrates for HRP include chromogenic substrates, 3,3’,5,5’-Tetramethylbenzidine, 3,3'-Diaminobenzidine and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid), and chemiluminescent substrates such as luminol. Examples of commonly used substrates for AP include chromogenic substrates, 4- nitrophenyl phosphate and 4-methylumbelliferyl phosphate. Biotin-streptavidin binding may similarly be detected.

[0028] As will be appreciated by one of skill in the art, other methods may be used to detect the target autoantibodies in a sample. For example, microparticle enzyme immunosorbent assay (MEIA) is a technique that utilizes very small microparticles in liquid suspension as a solid-phase support. Specific reagent antibodies are covalently bound to the microparticles. Antigen or target protein (e.g. actin, connexin-43, keratin or antigenic fragments thereof) is then bound to the immobilized antibody. Sample is added to the microparticles, and target autoantibodies, if present, bind to the antigen. Binding of the target antibodies is detected using an enzyme-based detection reaction in which enzyme is bound to the target autoantibodies and fluorescence is detected on addition of enzyme substrate to the reaction microparticle mix.

[0029] Latex agglutination may be used in which latex particles are coated with antigen (i.e. the target proteins or antigenic fragments thereof). Sample is added to the latex particles. If the target autoantibodies are present, they will bind with the corresponding antigens resulting in agglutination of the latex particles. Since there are three different target proteins, each one must be individually detected using this method to confirm the presence of each autoantibody.

[0030] An antibody sensor platform, known as LUMinescent AntiBody Sensor

(LUMABS) may also be used. It is based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution. LUMABS are single protein sensors that consist of the blue-light emitting luciferase, NanoLuc, connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to the target epitope sequence flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green- blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Again, as there are three different target proteins, each one must be individually detected to confirm the presence of each autoantibody.

[0031] Detection of the target autoantibodies in a mammalian sample is indicative of Brugada syndrome in the mammal. In addition, the level of autoantibodies correlates with disease burden to enable a determination of the classification, diagnostic category or confidence of disease, e.g. no Brugada vs. possible, borderline or definite Brugada. Thus, the greater the level of one or more of the target autoantibodies, the greater the disease burden.

[0032] The level of the target autoantibodies correlates with the intensity of signal generated by the detection method, e.g. the optical density using ELISA, band intensity using Western blotting, etc. For example, using ELISA, the optical density cut-point between no Brugada and borderline Brugada is about 0.05, 0.07, 0.08, 0.10, 0.12, 0.13 or higher, e.g. 0.1 - 0.12. In one embodiment, Brugada syndrome may be identified by the presence of antibody to specific actin, connexin-43 and keratin epitopes at various sample dilutions from at least about 1 :50 or greater, depending on the nature of the sample. For example, a dilution of at least about 1 :50 or greater is applicable for saliva or other non serological samples, while a dilution of at least about 1 : 100 is applicable for a serological sample, such as a dilution in the range of 1 : 100 to 1 : 1000. [0033] Thus, the present method provides a novel test, e.g. a serological test, for diagnosis of Brugada syndrome that is both highly sensitive (e.g. exhibits a sensitivity of greater than 75%, 80%, 85%, 90% or greater) and specific (e.g. exhibits specificity of greater than 75%, 80%, 85%, 90% or greater), and can readily be adapted for clinical diagnosis and the prediction of disease.

[0034] A mammal diagnosed with Brugada syndrome based on the presence of autoantibodies to actin, connexin-43 and keratin may be treated with a therapy targeted against at least one of the autoantibodies to inactivate one or more of the autoantibodies. The therapy may include treatment with a compound such as a protein, peptide, small molecule or antibody, that binds to one or more of the target autoantibodies and inactivates, at least partially, the autoantibody. The compound may be used alone or conjugated with an entity that assists to inactivate the autoantibody.

[0035] In one such embodiment, for example, immune cells such as T-cells, may be engineered to express a chimeric autoantibody receptor (CAAR) comprising an autoantigen, e.g. actin, connexin-43, keratin or an antigenic fragment(s) thereof and a cytoplasmic signaling domain. Antigenic fragments specific for the target autoantibodies comprise at least about 10-50 amino acids from an antigenic region of a target protein. As one of skill in the art will appreciate, the antigenic fragment may include consecutive amino acids from two regions, either in full or in part, or may include consecutive amino acids from one of these regions in full or in part. The autoantigen is fused to a transmembrane domain (e.g. dimerization-competent CD8a) and cytoplasmic signaling domain such as Oϋ137-Oϋ3z. Such engineered T-cells target cells that specifically bind to and inactivate the target proteins, namely, the target autoantibodies.

[0036] A mammal diagnosed with Brugada syndrome may also be treated in accordance with other embodiments of the present invention. Treatment options vary from mammal to mammal, and are based on the type of mammal, cardiac test results, medical history, and the presence or absence of genetic mutations. Treatment generally includes the use of an implantable cardioverter defibrillator and/or catheter ablation, including epicardial catheter or surgical ablation. Medications may additionally be administered to treat BrS, generally as an adjunct to device therapy or as an alternative when device therapy is not appropriate. For example, medications used may be aimed at rebalancing the currents active during phase 1 of the right ventricular action potential to abort electrical storms. In this regard, Class IA anti-arrhythmics such as quinidine, procainamide and disopyramide inhibit transient outward current and help to suppress ventricular tachycardia/fibrillation (VT/VF), and b-Adrenergic agonists such as isoproterenol and phosphodiesterase III inhibitors such as cilostazol function to boost calcium channel current.

[0037] Embodiments of the invention are described by reference to the following specific examples, which are not to be construed as limiting.

Examples

Example 1 - Identification of biomarkers associated with BrS

[0038] Serum from several subjects with Brugada syndrome (BrS), having a

Shanghai score of > 3.5 (probable and/or definite Brugada syndrome (BrS), possible BrS, and non-diagnostic outcomes were assigned scores of >3.5, based on the available published reports and on weighted coefficients derived from limited datasets) were assessed and compared to 5 control sera from normal individuals or family members with no Shanghai criteria. To identify anti-cardiac antibodies characterizing BrS, a modified technique of 2-Dimensional Gel Electropheresis (2-D Gel) was used to provide an array of cardiac proteins for the assessment of serum antibodies.

[0039] Using a method as illustrated in Figure 4, samples of normal human ventricular myocardium were homogenized, solubilized and separated based on isoelectric pH using an isoelectric focusing strip, such that each protein settled at its isoelectric point. Following this, the electropheresed proteins from the strip were transferred to a 2- dimensional gel and separated by molecular weight using standard electopheresis. These 2D gels were exposed to human sera in a 1 : 100 dilution, following which they were developed with a horseradish peroxidase linked anti-human IgG antibody. Autoantibody binding proteins were identified by scraping the spot off the gel and analyzing by mass spectrometry. Identified antibodies against specific cardiac proteins were then confirmed individually from BrS and control sera using individual Western blots.

[0040] The findings confirm a unique autoantibody profile to four protein spots of low molecular weight and high isoelectric pH in all four BrS sera, which are absent in control sera. Following evaluation of five proteins of appropriate size and isoelectric pH identified through mass spectrometry analysis of these 2D Gel spots, three proteins were identified (one with 2 isoforms) to which serum antibodies were binding and these were confirmed by Western blot as shown in Fig. 5.

[0041] The confirmatory Western blot analysis was performed with the sera at

1 : 100 dilutions using commercial recombinant a-cardiac actin, a-skeletal actin, keratin-24 and connexin-43 proteins. All recombinant proteins were purchased from Creative Biomart, USA.

[0042] A consistent autoantibody signature was identified against 4 proteins, namely actin (both isoforms, ACTA1 and ACTC1), connexin-43 and keratin-24, in 4 of 4 Brugada sera and 0 of 8 control sera. (p=0.0046. Fisher exact test). These results are significant even after Bonferonni adjustment for 5 comparisons (p<0.01). These findings have also been confirmed in 12 of 12 patient sera from a validation cohort of 12 additional Brugada subjects.

[0043] Autoantibody Assessments by ELISA - A direct Enzyme-Linked Immuno-

Sorbent Assay (ELISA) was performed by first coating a micro-titre plate with a-cardiac actin, a-skeletal actin, keratin and connexin-43 proteins according to the abeam protocol. For each well, 100 ng of protein in 100 mΐ of bicarbonate/carbonate coating buffer (100 mM sodium carbonate, pH 9.6) was placed and blocked with 200 mΐ of protease free BSA (2mg/ml; Cat # A3059; Sigma, USA). The wells were incubated with diluted human sera (100 mΐ of 1 : 100 dilution) for 2 hours at room temperature. Followed by washing the wells were incubated with anti-human IgG-HRP (cat # ab6759; abeam, USA) for 2 hours at room temperature. The resultant bound antibody was then assayed using Tetramethylbenzidine (TMB) chromophore to measure optical density at 450 nm wavelength. ELISAs of BrS serum from the discovery cohort, the validation cohort and controls detected antibodies against each protein (a-cardiac actin, a-skeletal actin, keratin-24 and connexin-43; Figure 6). In each case, ELISA optical densities from the discovery and validation cohort were equivalent, and markedly increased over the baseline optical densities as seen in control samples. These differences were statistically significant, and did not change for any group comparisons, even after computing the Hodges-Lehmann estimator for the group differences. [0044] Immunofluorescence Staining of Myocardial Tissues - To examine whether

BrS patient sera bind to the target proteins in cardiac tissue, normal cardiac tissue was double stained with BrS patient sera and commercial antibodies against a-cardiac actin, keratin-24 and connexin-43. Co-localization of staining patterns of BrS serum and all the commercial antibodies clearly demonstrated co-staining of a-cardiac actin, keratin-24 and connexin-43. Myocardium from a BrS decedent and biopsies from 9 BrS subjects were assessed. Each protein demonstrated abnormal aggregates within the sarcoplasm of BrS myocardium, as compared to normal tissue where a-cardiac actin expressed as filaments, and keratin-24 and connexin-43 demonstrated fine speckled staining. Sodium channel protein type 5 subunit alpha demonstrated similar large aggregates of staining within BrS cardiomyocytes. These aggregates were most convincing for keratin-24 and the cardiac sodium channel, and particle size analysis for both of these proteins separated Brugada decedent/biopsy patients from controls with 89% sensitivity.

Example 2 - Determination of Risk of BrS Associated with Autoantibodv levels

[0045] Participants & Recruitment: To evaluate risk associated with the level of autoantibodies, BrS subjects having a Shanghai score of > 3.5 are assessed. For these subjects, risk predictors, including total Shanghai score, are obtained and linked to serum sample. Based on data from collaborators, serum samples have been collected on 2149 subjects, of which 1/3 will have had events. The ability of each of the 3 identified autoantibodies is assessed to determine which one is most predictive of prior events.

[0046] Each autoantibody of the BrS Ab profile is assessed to determine correlation with risk, such as total Shanghai score. All risk predictors are assessed including each autoantibody by univariate analysis to determine if they predict adverse events, e.g. sudden cardiac arrest (SCA) or death (SCD) or appropriate implantable cardioverter-defibrillator (ICD) discharge. Significant predictors will then be entered into a multivariate model. Given that not every appropriate ICD discharge represents an aborted SCD, this outcome is weighted by 50%.

[0047] An initial cross-sectional analysis of antibody level will be determined (by

Western density and ELISA optical density), and these levels will be correlated with accepted measures of disease severity and risk. These include age, sex, prior SCA, prior syncope, genotype, proband status, spontaneous versus drug-induced Brugada ECG pattern, right ventricular outflow tract dilation or dysfunction, extent of T-wave inversion and (where available) response to programmed ventricular stimulation. The correlation of antibody levels with adverse events at 5 years of follow-up are also assessed.

Example 3

[0048] The presence of antibodies to Actin, Connexin-43 and Keratin in the sera of subjects with Brugada Syndrome, i.e. circulating autoantibodies, which are not detected in the sera of normal individuals, indicates that the complex connexome network in the intercalated disk may become disrupted in Brugada syndrome, releasing these proteins such that epitopes not normally exposed to antigen presenting cells initiate an autoimmune response. Since these proteins, particularly keratin, exist as a broad range of similar proteins with substantial homology, the target antigenic epitope for each is determined.

[0049] Immunohistology: The ultrastructure of human right ventricular outflow tract myocardium from Brugada victims (via postmortem retained tissue blocks) and Brugada subjects (via endomyocardial biopsy specimens from the right ventricular outflow tract) is compared to equivalent tissues from normal individuals. Postmortem specimens (Brugada and control) were kindly provided as a collaboration with Dr. Kris Cunningham, Medical Director of the Ontario Provincial Forensic Pathology Unit and biopsy specimens were provided by Dr. Maurizio Pieroni of Cardiologo San Donato Hospital, Arezzo, Italy. These specimens are examined by super-resolution microscopy techniques using immunohistochemical staining against Actin, Connexin-43 and Keratin, separately and in combination, to determine if ultrastructural differences occur in the expression of these proteins in cardiomyocytes of the right ventricular outflow tract. In addition, subsequent specimens will be assessed by electron microscopy, including multi-particle gold immune- electron microscopy and tomographic electron microscopy to assess ultrastructural changes in the intercalated disk.

[0050] Epitope mapping: Linear epitope mapping is conducted to determine the specific autoimmune epitopes on Actin, Connexin-43 and Keratin targeted by autoantibodies in Brugada Syndrome. Linear epitope libraries are provided by Thermo Fisher Scientific: Pierce Protein Research Products and consist of 15 amino acid oligopeptides of the entire length of the respective protein, with 5 amino acids of overlap. Identification of the target epitope for each protein provides an assay that retains sensitivity but which may enhance specificity.

[0051] Functional studies: Using sera from Brugada subjects, control sera and commercial sources of Actin, Connexin-43 and Keratin antibodies, the gap junction dysfunction in confluent cultured cardiomyocytes will be assessed to determine if the Brugada serum autoantibodies result in significant reduction in gap junction function, resulting on the observed phenomenon of conduction delay in BrS. Confirmation that the autoantibodies affect gap junction function indicates that they are a therapeutic target.

Claims

1. A method of detecting target autoantibodies to actin, connexin-43 and keratin in a mammal comprising:

2) detecting the presence of the target autoantibodies in the sample by detecting binding of the target autoantibodies to each of the antigens.

2. The method of claim 1, wherein the actin comprises alpha cardiac muscle actin (ACTC1), alpha skeletal muscle actin (ACTA1) or a combination thereof.

3. The method of claim 1, wherein the keratin is keratin-24.

4. The method of claim 1, wherein the actin, keratin and connexin-43 are human proteins.

5. The method of claim 1, wherein an antigenic fragment of actin is used comprising a sequence of at least about 10 amino acids from SEQ ID NO: 1 or SEQ ID NO: 2.

6. The method of claim 1 , wherein an antigenic fragment of keratin is used comprising a sequence of at least about 10 amino acids from SEQ ID NO: 3.

7. The method of claim 1, wherein an antigenic fragment of connexin-43 is used comprising a sequence of at least about 10 amino acids from SEQ ID NO: 4.

8. The method of claim 1, wherein the antigens or antigenic fragments are bound to a solid support.

9. The method of claim 1, wherein bound target autoantibodies are detected using labelled secondary antibody.

10. The method of claim 9, wherein the secondary antibody is labelled with an enzyme label, a fluorescent label, an affinity label or a radioactive label.

11. The method of claim 1, which is an ELISA.

12. The method of claim 1, which is a Western blotting method.

13. The method of claim 1, wherein the biological sample is a serological sample.

14. A method of diagnosing Brugada syndrome in a mammal comprising the steps of:

1) contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin antigens, or with antigenic fragments thereof, to bind with the target autoantibodies to the antigens;

2) detecting the presence of target autoantibodies in the sample by detecting binding of the target autoantibodies to each of the antigens; and

15. The method of claim 14, wherein the actin comprises alpha cardiac muscle actin (ACTC1), alpha skeletal muscle actin (ACTA1) or a combination thereof.

16. The method of claim 14, wherein the keratin is keratin-24.

17. The method of claim 14, wherein the actin, keratin and connexin-43 are human proteins.

18. The method of claim 14, wherein an antigenic fragment of actin is used comprising a sequence of at least about 10 amino acids from SEQ ID NO: 1 or SEQ ID NO: 2.

19. The method of claim 14, wherein an antigenic fragment of keratin is used comprising a sequence of at least about 10 amino acids from SEQ ID NO: 3.

20. The method of claim 14, wherein an antigenic fragment of connexin-43 is used comprising a sequence of at least about 10 amino acids from SEQ ID NO: 4.

21. The method of claim 14, wherein the antigens or antigenic fragments are bound to a solid support.

22. The method of claim 14, wherein bound target autoantibodies are detected using labelled secondary antibody.

23. The method of claim 22, wherein the secondary antibody is labelled with an enzyme label, a fluorescent label, an affinity label or a radioactive label.

24. The method of claim 14, which is an ELISA.

25. The method of claim 14, which is a Western blotting method.

26. The method of claim 14, wherein the biological sample is a serological sample.

27. A method of diagnosing and treating Brugada syndrome in a mammal comprising the steps of:

1) contacting a biological sample obtained from the mammal with each of actin, connexin-43 and keratin antigens, or with antigenic fragments thereof, to bind with the target autoantibodies;

2) detecting the presence of the target autoantibodies in the sample by detecting binding of the target autoantibodies to each of the antigens;

3) diagnosing the mammal with Brugada syndrome when the presence of the target autoantibodies is detected; and

4) treating the mammal with one or more of an implantable cardioverter defibrillator, catheter ablation and medication.

28. The method of claim 27, wherein the medication is selected from a therapy that rebalances the currents active during phase 1 of the right ventricular action potential to abort electrical storms and to boost calcium channel current.

29. The method of claim 27, wherein the medication is selected from Class IA antiarrhythmics, b-adrenergic agonists and phosphodiesterase III inhibitors.