KR20220034725A - Detection of biomarkers associated with Brugada syndrome - Google Patents

Abstract

Methods are provided for detecting target autoantibodies to actin, connexin-43 and keratin in a mammal. The method comprises contacting a biological sample obtained from a mammal with each of actin, connexin-43 and keratin or an antigenic fragment thereof; and detecting the presence of the target autoantibody in the sample by detecting binding of the target autoantibody to the respective antigen. The method is useful for diagnosing Brugada syndrome.

Description

Detection of biomarkers associated with Brugada syndrome

FIELD OF THE INVENTION The present invention relates generally to Brugada Syndrome, and in particular to a method of diagnosing Brugada Syndrome.

Brugada syndrome (BrS) is an inherited autosomal dominant disorder with abnormal electrical activity of the heart. BrS and a related condition called 'early repolarization syndrome (ERS)' are together called 'J-wave syndrome'. In Southeast Asia, it is referred to as 'Sudden Unexpected Nocturnal Death Syndrome', in Thailand 'Lai Tai' (dreaming and screaming death), in Vietnam 'Tsob Tsuang' (Nightmare Death Syndrome), in the Philippines ' Bangungut' (waking up and moaning during sleep), 'Phi Am' (widow ghost) in Taiwan, 'Pokkuri' ('die') in Japan, 'Sudden Death' in Korea, 'Dream Disease' in Hawaii and 'Sudden death of young adults' in mainland China Syndrome is called locally.

BrS is characterized by a coved-type ST-segment elevation in the right precordial lead of an electrocardiogram (ECG), although the appearance of this feature is often transient. The Brugada ECG pattern is typically observed in standard leads V1-V3 in the anterior thoracic lead above the right ventricular outflow tract (RVOT), or in a modified lead position placed higher in the chest. Consistent with this local abnormality, some studies report inflammation as well as enlargement in this area. In addition, consistent with local abnormalities, atypical electrocardiograms are found on the epicardial surface of RVOT, and epicardial resection of this region can prevent ventricular fibrillation episodes associated with Brugada syndrome and normalize ECG in most patients. . BrS is associated with a relatively high risk of sudden death in young adults (especially men), and sometimes in children and infants. The event is exacerbated by fever, alcohol, overeating, high vagal tone, and certain medications.

The prevalence of BrS is estimated to be 1:2000, or greater than about 18,000, in Canada. It is much more prevalent among peoples of Southeast Asia. The study showed a prevalence of 0.05 - 0.6% ECG compatible with Brugada syndrome in regions such as Japan. Brugada syndrome accounts for 4%-12% of unexpected and sudden deaths and up to 20% of all sudden deaths in individuals with a seemingly normal heart. In Southeast Asia, it is the second-highest number of deaths among men under the age of 40 (after accidents).

Because of the lack of a simple and accurate test for BrS, the majority of an estimated 18,000 affected individuals in Canada have not been diagnosed, evaluated, or managed. Clinical confirmation is often transient or depends on a specific electrocardiogram pattern requiring drug provocation. Genetic diagnosis does not identify 75% of affected patients/family. Mutations in the SCN5A sodium channel have been identified in 25% of patients, but other genetic causes have no acceptable evidence for a causal relationship. Identifying individuals at risk of developing the disease is difficult. The lack of simple and accurate tests that can identify preclinical disease makes cascade screening in BrS (a key tool in other genetic arrhythmic conditions) difficult. Although the main symptoms identify a small group of high-risk individuals and the spontaneous type 1 Brugada ECG pattern identifies mild risk, risk stratification in BrS patients is incomplete.

Therefore, it would be desirable to develop a simple and sensitive test sufficient to diagnose BrS.

Unique autoantibody signatures for specific proteins associated with Brugada syndrome have now been identified, and their determination is therefore useful for the diagnosis and, optionally, treatment of BrS in mammals.

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

1) contacting a biological sample obtained from a mammal with actin, connexin-43 and keratin antigens, respectively, or antigen fragments thereof to bind the target autoantibody; and

2) detecting the presence of the target autoantibody in the sample by detecting binding of the target autoantibody to the respective antigen.

In another aspect of the invention, a method of diagnosing Brugada syndrome in a mammal is provided. The method comprises the following steps:

1) contacting a biological sample obtained from a mammal with actin, connexin-43 and keratin antigens, respectively, or antigen fragments thereof, to bind autoantibodies to the respective antigens;

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

3) diagnosing the mammal as having Brugada syndrome when the presence of the target autoantibody is detected.

In a further aspect of the invention, there is provided a method of diagnosing and treating Brugada syndrome in a mammal. The method comprises the following steps:

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

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

3) diagnosing the mammal as having Brugada syndrome when the presence of each target autoantibody is detected; and

4) treating the mammal with one or more of an implantable defibrillator, catheter ablation and a drug selected from a class IA antiarrhythmic agent, a β-adrenergic agonist and a phosphodiesterase III inhibitor.

These and other aspects of the invention will become apparent upon reference to the following detailed description.

1 illustrates the amino acid sequences of human alpha 1 myocardial actin and human alpha 1 skeletal muscle actin, respectively;
2 illustrates the amino acid sequence of keratin 24;
3 illustrates the amino acid sequence of connexin-43;
4 is a schematic diagram illustrating a method for identifying Brugada autoantibodies;
5 illustrates the results of Western blot analysis for Brugada Syndrome samples and healthy control samples;
6 is an enzyme-linked immunosorbent demonstrating antibodies to (A) connexin-43, (B) cardiac α-actin, (C) skeletal α-actin, and (D) keratin-24 in discovery and validation cohorts versus controls. The results of the assay (ELISA) are illustrated graphically.

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

A biological sample obtained from a 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 bodily fluids that can be obtained non-invasively. The biological sample can be obtained using methods well established in the art, can be obtained directly from the mammal, or previously from a mammal that has been properly stored for later use (eg, stored at 4°C). It can be obtained from the obtained sample. A sample amount of at least about 100 μl, eg, 100 μl, of diluted human serum (1:100 dilution in blocking buffer) can be used to perform the methods of the present invention.

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.

The sample is contacted with actin, connexin-43 and keratin, respectively, comprising the full-length version of the protein or antigenic fragment thereof.

Actin is used herein to refer to mammalian actin, and in particular the isoforms, alpha-myocardial 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 myocardial actin and human alpha 1 skeletal muscle actin are shown in Figures 1A) and B), respectively. Functionally equivalent isoforms and variants comprising amino acid replacements that do not affect function, such as variant proteins from different species, are also included. The term "functionally equivalent" refers to isotypes and variants that retain the function of the wild-type protein, but not necessarily to the same extent, but at least partially, such as retaining at least about 25% or more of the activity of the wild-type protein. An antigenic region of actin, such as a region comprising an antibody binding site, can be used to prepare antigenic fragments of actin for use in the methods of the present invention. In one embodiment, antigenic fragments can be prepared from within 30 amino acids of the N-terminal region of actin, such as 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. Antigen fragments can be of various sizes, such as 12-20 amino acids or more, such as 30, 40, 50 or more amino acids.

Connexin-43, also referred to as gap junction alpha-1 protein (GJA1), is a neurotransmitter that connects neighboring cells that allows the exchange of low molecular weight molecules such as small ions and secondary messengers to maintain homeostasis. It is a component of the gap junction, which is an intracellular channel. The protein comprises N- and C-terminal regions, and multiple transmembrane domains. As used herein, connexin-43 includes mammalian proteins. The amino acid sequence of human connexin-43 is shown in FIG. 3 . Functionally equivalent isoforms and variants comprising amino acid replacements that do not affect function, such as variant proteins from different species, are also included. The antigenic region of connexin-43 comprises, for example, the first 230 amino acids of the C-terminus of the protein.

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, and the like. The target keratin may be a cardiac expressed keratin, including but not limited to type 1 (acidic) keratins such as keratins 8, 18, 23 and/or 24. The amino acid sequence of human keratin 24 is shown in FIG. 2 . Functionally equivalent isoforms and variants containing amino acid replacements that do not affect function, such as variant keratin proteins from different species, are also included. An antigenic region of keratin, such as a region comprising an antibody binding site, can be used to prepare antigenic fragments of keratin for use in the methods of the invention. For example, the antigenic region may comprise, in whole or in part, amino acids from positions 120-400 of the keratin.

Actin, connexin-43 and keratin antigens, or antigenic fragments thereof, are preferably bound to a solid support such as nitrocellulose, polyvinylidene difluoride (PVDF), or cationic nylon membrane to capture the target autoantibody, or is fixed As is known to those skilled in the art, to prevent non-specific binding on a solid support, free binding sites on the support are blocked using a suitable blocking buffer, such as milk, normal serum or purified protein. The assay utilizes conditions suitable for autoantibody binding to the protein, eg, incubation at an appropriate temperature in a suitable buffer, for example. After sufficient time for binding to occur, the solid support is washed to remove unbound and/or non-specifically bound material. Physiological buffers such as Tris Buffered Saline (TBS) or Phosphate Buffered Saline (PBS) may be used, optionally including additives such as detergents (eg, 0.05% Tween ^™ 20).

The presence of the target autoantibody in the sample is then determined by detecting the autoantibody that binds to the target actin, connexin-43 and keratin protein, or bound to an antigenic fragment thereof. Methods such as Western blotting or immunoassays such as enzyme linked immunoassay (ELISA) can be used for this purpose. Other antibody detection methods may also be used.

In one embodiment, the bound target autoantibody binds to the target autoantibody and is detected using a detectable secondary antibody. If the sample is a human sample, a non-human mammal (eg, goat, rabbit, mouse, rat, chicken, pig, cow, sheep) directed against an anti-human secondary antibody, eg, a human immunoglobulin such as immunoglobulin G (IgG). , donkey) can be used. If the sample is a non-human sample, a suitable secondary antibody, obtainable from a different non-human mammal, is used to detect the target autoantibody. To detect bound target autoantibody, the sample is contacted with a secondary antibody under conditions suitable for binding and then washed to remove unbound reagent. The secondary antibody is labeled with any suitable detectable label either before or after autoantibody binding using established protocols. Suitable labels include, but are not limited to, enzymatic labels such as glucose oxidase, horseradish peroxidase (HRP) or alkaline phosphatase (AP); fluorescent labels such as ethidium bromide, fluorescein, rhodamine, phycoerythrin, cyanine, coumarin, green fluorescent protein and derivatives thereof; affinity labels such as biotin/streptavidin labels; or a radioactive label. The presence of the target autoantibody is then detected by detecting the presence of the selected label using methods known to those skilled in the art. For example, to detect an enzyme label, an appropriate enzyme substrate is added to the sample and the enzyme activity is detected by chromogenic, chemiluminescent or fluorescence output. Examples of commonly used substrates for HRP are the chromogenic substrates, 3,3',5,5'-tetramethylbenzidine, 3,3'-diaminobenzidine and 2,2'-azino-bis(3-ethylbenzo thiazoline-6-sulfonic 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 can be detected similarly.

As will be appreciated by one of ordinary skill in the art, other methods may be used to detect the target autoantibody in a sample. For example, microparticle enzyme immunosorbent assay (MEIA) is a technique that uses very small microparticles in liquid suspension as solid phase supports. Certain reagent antibodies are covalently bound to microparticles. The antigen or target protein (eg, actin, connexin-43, keratin or antigen fragment thereof) is then bound to the immobilized antibody. A sample is added to the microparticles and the target autoantibody, if present, binds to the antigen. Binding of the target antibody is detected using an enzyme-based detection reaction in which the enzyme is bound to the target autoantibody and fluorescence is detected upon addition of the enzyme substrate to the reaction microparticle mixture.

Latex aggregation in which latex particles are coated with an antigen (ie, a target protein or antigenic fragment thereof) can be used. A sample is added to the latex particles. If the target autoantibodies are present, they will bind the corresponding antigen and result in aggregation of the latex particles. Since there are three different target proteins, each must be detected individually using this method to confirm the presence of each autoantibody.

Antibody sensor platforms, also known as LUMinescent AntiBody Sensors (LUMABS), may also be used. It is based on bioluminescence resonance energy transfer (BRET), which can detect antibodies directly in solution. LUMABS is a single protein sensor composed of NanoLuc, a blue-emitting luciferase, linked to the green fluorescent receptor protein mNeonGreen via a semi-flexible linker, which are held close to each other using helper domains. Binding of the antibody to the target epitope sequence flanking the linker disrupts the interaction between the helper domains, greatly reducing BRET efficiency. The resulting color change of emitted light from green-blue to blue can be detected directly in plasma even at picomolar concentrations of the antibody. In addition, since there are three different target proteins, each must be detected individually using this method to confirm the presence of each autoantibody.

Detection of target autoantibodies in mammalian samples is indicative of Brugada syndrome in mammals. In addition, the level of autoantibody correlates with disease class, diagnostic category or reliability, such as no Brugada versus disease burden that allows for the determination of probable, borderline or definitive Brugada. Thus, the higher the level of one or more target autoantibodies, the greater the disease burden.

The level of the target autoantibody correlates with the intensity of the signal generated by the detection method, such as optical density using ELISA, band intensity using Western blotting, and the like. For example, using ELISA, the optical density cutpoints between no Brugada and borderline Brugada are at least about 0.05, 0.07, 0.08, 0.10, 0.12, 0.13, such as 0.1 - 0.12. In one embodiment, Brugada syndrome can be identified by the presence of antibodies to specific actin, connexin-43 and keratin epitopes at various sample dilutions of 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 to saliva or other non-serological samples, whereas a dilution of at least about 1:100, such as a dilution ranging from 1:100 to 1:1000, is applicable to a serological sample. Do.

Thus, the methods of the present invention are highly sensitive (e.g., exhibiting a sensitivity of at least 75%, 80%, 85%, 90%) and specific (e.g., having a specificity of at least 75%, 80%, 85%, 90%). ), provides a novel test for the diagnosis of Brugada syndrome, such as a serological test, which can be easily applied to clinical diagnosis and disease prediction.

Mammals diagnosed with Brugada syndrome based on the presence of autoantibodies to actin, connexin-43 and keratin may be treated with a targeted therapy to at least one of the autoantibodies to inactivate one or more of the autoantibodies. there is. 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 at least partially inactivates the autoantibodies. Compounds can be used alone or conjugated with entities that help inactivate autoantibodies.

In one such embodiment, for example, an immune cell, such as a T-cell, is a chimeric autologous cell comprising an autoantigen such as actin, connexin-43, keratin or antigen fragment(s) thereof and a cytoplasmic signaling domain. It can be engineered to express an antibody receptor (CAAR). An antigenic fragment specific for a target autoantibody comprises at least about 10-50 amino acids from the antigenic region of the target protein. As will be appreciated by those of skill in the art, an antigenic fragment may comprise in whole or in part conservative amino acids from two regions, or may comprise in whole or in part conservative amino acids from one of these regions. The autoantigen is fused to a transmembrane domain (eg, dimerizable CD8α) and a cytoplasmic signaling domain such as CD137-CD3ζ. These engineered T cells target a target protein, ie, a cell that specifically binds to and inactivates the target autoantibody.

Mammals diagnosed with Brugada syndrome may also be treated according to other embodiments of the invention. Treatment options vary from mammal to mammal and are based on the type of mammal, cardiac examination results, medical history, and the presence or absence of genetic mutations. Treatment generally involves the use of an epicardial catheter or an implantable defibrillator including surgical ablation and/or catheter ablation. In general, additional drugs may be administered to treat BrS as an adjunct to device therapy or as an alternative when device therapy is not appropriate. For example, the drug used may aim to halt the electrical storm by rebalancing the current that is active during phase 1 of the right ventricular action potential. In this regard, class IA antiarrhythmic agents such as quinidine, procainamide and diisopyramide help to suppress transient outward currents and suppress ventricular tachycardia/fibrillation (VT/VF), and β-adrenergic agonists such as Isoproterenol and phosphodiesterase III inhibitors, such as cilostazol, function to increase calcium channel currents.

Embodiments of the invention are described with reference to the following specific examples, which should not be construed as limiting.

Example

Example 1 - Identification of biomarkers related to BrS

Serum from several subjects with Brugada Syndrome (BrS), with a Shanghai score of ≥ 3.5 (probable and/or definitive Brugada Syndrome (BrS), possible BrS, and non-diagnostic results available, published assigned a score of ≧3.5 based on the report and based on weighted coefficients derived from a limited dataset) and compared to 5 control sera from normal individuals or family members without the Shanghai criterion. To identify anti-cardiac antibodies characterizing BrS, a modified technique of two-dimensional gel electrophoresis (2-D Gel) was used to provide a series of cardiac proteins for the evaluation of serum antibodies.

Using the method as illustrated in Figure 4, samples of normal human ventricular myocardium were homogenized, solubilized and separated based on isoelectric point pH using an isoelectric focusing strip such that each protein settles at its isoelectric point. The electrophoresed proteins from the strips were then transferred to a two-dimensional gel and separated by molecular weight using standard electrophoresis. These 2D gels were exposed to 1:100 diluted human serum and then developed with horseradish peroxidase linked anti-human IgG antibody. Autoantibody binding proteins were identified by scraping spots from the gel and analyzing by mass spectrometry. Antibodies identified against specific cardiac proteins were then individually identified from BrS and control sera using separate Western blots.

This finding confirms a unique autoantibody profile against four protein spots of low molecular weight and high isoelectric pH in all four BrS sera, which are not present in control sera. After evaluating 5 proteins of appropriate size and isoelectric point pH identified through mass spectrometry analysis of these 2D gel spots, 3 proteins to which serum antibodies were bound were identified (one with 2 isoforms), and these were shown in Fig. It was confirmed by Western blot as shown in Fig.

Confirmatory Western blot analysis was performed with serum diluted 1:100 using commercial recombinant α-cardiac actin, α-skeletal actin, keratin-24 and connexin-43 proteins. All recombinant proteins were purchased from Creative Biomart, USA.

In 4 of 4 Brugada sera and 0 of 8 control sera, consistent autoantibody signatures were identified for 4 proteins: actin (two isoforms, ACTA1 and ACTC1), connexin-43 and keratin-24. (p=0.0046. Fisher's exact test). These results are significant even after Bonferroni adjustment for 5 comparisons (p<0.01). These findings were also confirmed in 12 of 12 patient sera from a validation cohort of 12 additional Brugada subjects.

Autoantibody Assessment by ELISA - Direct enzyme-linked immunosorbent assay (ELISA) was performed by first coating microtiter plates with α-cardiac actin, α-skeletal actin, keratin and connexin-43 proteins according to the abcam protocol. For each well, add 100 ng of protein in 100 μl of bicarbonate/carbonate coating buffer (100 mM sodium carbonate, pH 9.6) and 200 μl of protease-free BSA (2 mg/ml; Cat # A3059; Sigma, USA). Wells were incubated with diluted human serum (1 :100 dilution of 100 μl) for 2 h at room temperature. After washing the wells, they were incubated with anti-human IgG-HRP (cat # ab6759; abcam, USA) for 2 hours at room temperature. Then, the resulting bound antibody was analyzed using a tetramethylbenzidine (TMB) chromophore to measure the optical density at a wavelength of 450 nm. ELISA of BrS sera from discovery cohorts, validation cohorts and controls detected antibodies to each protein (α-cardiac actin, α-skeletal actin, keratin-24 and connexin-43; FIG. 6 ). In each case, the ELISA optical densities from the discovery and validation cohorts were equivalent and significantly increased over baseline optical densities as seen in the control samples. These differences were statistically significant and did not change for any group comparisons after calculating the Hodges-Lehmann estimator for group differences.

Immunofluorescent staining of myocardial tissue - To investigate whether BrS patient sera binds to target proteins in cardiac tissue, normal cardiac tissue was subjected to BrS patient sera and commercial antibodies to α-cardiac actin, keratin-24 and connexin-43. was double-stained with Co-localization of staining patterns of BrS serum and all commercial antibodies clearly demonstrated co-staining of α-cardiac actin, keratin-24 and connexin-43. Myocardium from BrS deaths and 9 BrS subjects were evaluated. Each protein demonstrated aberrant aggregates within the sarcoplasm of BrS myocardium compared to normal tissue in which α-cardiac actin was expressed as filaments and keratin-24 and connexin-43 showed fine speckle staining. The sodium channel protein type 5 subunit alpha demonstrated similar large staining aggregates in BrS cardiomyocytes. These aggregates were most convincing for keratin-24 and cardiac sodium channels, and particle size analysis for these two proteins separated Brugada dead/biopsy patients with 89% sensitivity.

Example 2 - Determination of Risk of BrS Associated with Autoantibody Levels

Participants and Recruitment: To assess risk associated with levels of autoantibodies, BrS subjects with a Shanghai score of ≧3.5 are evaluated. For these subjects, risk predictors, including total Shanghai scores, are obtained and associated with serum samples. Based on data from collaborators, serum samples were collected for 2149 subjects, one-third of whom would have had an event. The ability of each of the three identified autoantibodies is evaluated to determine which one best predicts the previous event.

Each autoantibody in the BrS Ab profile is evaluated to determine its correlation with risk, such as the total Shanghai score. All risk predictors, including each autoantibody, were evaluated by univariate analysis to determine whether they predict adverse events, such as acute cardiac arrest (SCA) or death (SCD) or appropriate implantable defibrillator (ICD) discharge. do. Then, we will input the important predictors into the multivariate model. We weight this result by 50%, taking into account that not all appropriate ICD discharges are indicative of a stopped SCD.

An initial cross-sectional analysis of antibody levels will be determined (by Western density and ELISA optical density), which will correlate with accepted measures of disease severity and risk. These included age, sex, previous SCA, previous syncope, genotype, proband status, spontaneous versus drug-induced Brugada ECG pattern, right ventricular outflow tract dilatation or dysfunction, extent of T-wave inversion and (use if possible) in response to programmed ventricular stimulation. Correlation of antibody levels with adverse events at 5-year follow-up is also assessed.

Example 3

The presence of antibodies to actin, connexin-43, and keratin in the serum of subjects with Brugada syndrome, i.e., circulating autoantibodies that are not detected in the serum of normal individuals, is the result of a complex connexome network within the inserted disc. This indicates that epitopes that are destroyed in Garda syndrome and not normally exposed to antigen-presenting cells can release these proteins to initiate an autoimmune response. Since these proteins, particularly keratins, exist as a wide range of similar proteins with significant homology, the target antigenic epitope for each is determined.

Immunohistochemistry: The ultrastructure of human right ventricular outflow myocardium from Brugada victims (via post-mortem maintenance tissue blocks) and Brugada subjects (via endomyocardial biopsy specimens from the right ventricular outflow tract) was compared with equivalent tissues from normal individuals. Compare. Postmortem specimens (Brugada and control) were kindly provided in collaboration with Dr. Kris Cunningham, Medical Director, Department of Forensic Pathology, Ontario State, and biopsy specimens were obtained from Cardiologo San Donato Hospital, Arezzo, Italy. Donato Hospital, Dr. Maurizio Pieroni. Examination of these specimens individually and by super-resolution microscopy using immunohistochemical staining for actin, connexin-43 and keratin in combination resulted in microstructural differences in the expression of these proteins in cardiomyocytes of the right ventricular outflow tract. decide whether In addition, subsequent specimens will be evaluated by electron microscopy, including multi-particle gold immuno-electron microscopy and tomographic electron microscopy, to assess microstructural changes within the implanted disc.

Epitope Mapping: Linear epitope mapping is performed to determine specific autoimmune epitopes for actin, connexin-43 and keratin targeted by autoantibodies in Brugada syndrome. The linear epitope library is provided by Thermo Fisher Scientific: Pierce Protein Research Products and consists of 15 amino acid oligopeptides of the total length of each protein, with a 5 amino acid overlap. Identification of target epitopes for each protein provides an assay that can improve specificity while maintaining sensitivity.

Functional Study: Brugada serum autologous to assess gap junction dysfunction in confluent cultured cardiomyocytes using sera from Brugada subjects, control sera and commercial sources of actin, connexin-43 and keratin antibodies. It will be determined whether the antibody results in a significant decrease in gap junction function resulting in the conduction delay phenomenon observed in BrS. Confirmation that autoantibodies affect gap junction function indicates that they are therapeutic targets.

SEQUENCE LISTING <110> The Hospital for Sick Children <120> DETECTION OF BIOMARKERS ASSOCIATED WITH BRUGADA SYNDROME <130> H8313075PCT <140> PCT CA2020/050578 <141> 2020-05-01 <150> US 62/842124 <151> 2019-05-02 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 377 <212> PRT <213> Homo sapiens <400> 1 Met Cys Asp Asp Glu Glu Thr Thr Ala Leu Val Cys Asp Asn Gly Ser 1 5 10 15 Gly Leu Val Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val 20 25 30 Phe Pro Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly 35 40 45 Met Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg 50 55 60 Gly Ile Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Ile Thr Asn 65 70 75 80 Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu 85 90 95 Arg Val Ala Pro Glu Glu His Pro Thr Leu Leu Thr Glu Ala Pro Leu 100 105 110 Asn Pro Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr 115 120 125 Phe Asn Val Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu 130 135 140 Tyr Ala Ser Gly Arg Thr Thr Gly Ile Val Leu Asp Ser Gly Asp Gly 145 150 155 160 Val Thr His Asn Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala 165 170 175 Ile Met Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met 180 185 190 Lys Ile Leu Thr Glu Arg Gly Tyr Ser Phe Val Thr Thr Ala Glu Arg 195 200 205 Glu Ile Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp 210 215 220 Phe Glu Asn Glu Met Ala Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys 225 230 235 240 Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg 245 250 255 Phe Arg Cys Pro Glu Thr Leu Phe Gln Pro Ser Phe Ile Gly Met Glu 260 265 270 Ser Ala Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp 275 280 285 Ile Asp Ile Arg Lys Asp Leu Tyr Ala Asn Asn Val Leu Ser Gly Gly 290 295 300 Thr Thr Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr 305 310 315 320 Ala Leu Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu 325 330 335 Arg Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser 340 345 350 Thr Phe Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ala Gly 355 360 365 Pro Ser Ile Val His Arg Lys Cys Phe 370 375 <210> 2 <211> 377 <212> PRT <213> Homo sapiens <400> 2 Met Cys Asp Glu Asp Glu Thr Thr Ala Leu Val Cys Asp Asn Gly Ser 1 5 10 15 Gly Leu Val Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val 20 25 30 Phe Pro Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly 35 40 45 Met Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg 50 55 60 Gly Ile Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Ile Thr Asn 65 70 75 80 Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu 85 90 95 Arg Val Ala Pro Glu Glu His Pro Thr Leu Leu Thr Glu Ala Pro Leu 100 105 110 Asn Pro Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr 115 120 125 Phe Asn Val Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu 130 135 140 Tyr Ala Ser Gly Arg Thr Thr Gly Ile Val Leu Asp Ser Gly Asp Gly 145 150 155 160 Val Thr His Asn Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala 165 170 175 Ile Met Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met 180 185 190 Lys Ile Leu Thr Glu Arg Gly Tyr Ser Phe Val Thr Thr Ala Glu Arg 195 200 205 Glu Ile Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp 210 215 220 Phe Glu Asn Glu Met Ala Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys 225 230 235 240 Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg 245 250 255 Phe Arg Cys Pro Glu Thr Leu Phe Gln Pro Ser Phe Ile Gly Met Glu 260 265 270 Ser Ala Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp 275 280 285 Ile Asp Ile Arg Lys Asp Leu Tyr Ala Asn Asn Val Met Ser Gly Gly 290 295 300 Thr Thr Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr 305 310 315 320 Ala Leu Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu 325 330 335 Arg Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser 340 345 350 Thr Phe Gln Gln Met Trp Ile Thr Lys Gln Glu Tyr Asp Glu Ala Gly 355 360 365 Pro Ser Ile Val His Arg Lys Cys Phe 370 375 <210> 3 <211> 472 <212> PRT <213> Homo sapiens <400> 3 Met Thr Thr Cys Ser Arg Gln Phe Thr Ser Ser Ser Ser Met Lys Gly 1 5 10 15 Ser Cys Gly Ile Gly Gly Gly Ile Gly Gly Gly Ser Ser Arg Ile Ser 20 25 30 Ser Val Leu Ala Gly Gly Ser Cys Arg Ala Pro Ser Thr Tyr Gly Gly 35 40 45 Gly Leu Ser Val Ser Ser Ser Arg Phe Ser Ser Gly Gly Ala Cys Gly 50 55 60 Leu Gly Gly Gly Tyr Gly Gly Gly Phe Ser Ser Ser Ser Ser Ser Ser Phe 65 70 75 80 Gly Ser Gly Phe Gly Gly Gly Tyr Gly Gly Gly Leu Gly Ala Gly Leu 85 90 95 Gly Gly Gly Phe Gly Gly Gly Gly Phe Ala Gly Gly Asp Gly Leu Leu Val 100 105 110 Gly Ser Glu Lys Val Thr Met Gln Asn Leu Asn Asp Arg Leu Ala Ser 115 120 125 Tyr Leu Asp Lys Val Arg Ala Leu Glu Glu Ala Asn Ala Asp Leu Glu 130 135 140 Val Lys Ile Arg Asp Trp Tyr Gln Arg Gln Arg Pro Ala Glu Ile Lys 145 150 155 160 Asp Tyr Ser Pro Tyr Phe Lys Thr Ile Glu Asp Leu Arg Asn Lys Ile 165 170 175 Leu Thr Ala Thr Val Asp Asn Ala Asn Val Leu Leu Gln Ile Asp Asn 180 185 190 Ala Arg Leu Ala Ala Asp Asp Phe Arg Thr Lys Tyr Glu Thr Glu Leu 195 200 205 Asn Leu Arg Met Ser Val Glu Ala Asp Ile Asn Gly Leu Arg Arg Val 210 215 220 Leu Asp Glu Leu Thr Leu Ala Arg Ala Asp Leu Glu Met Gln Ile Glu 225 230 235 240 Ser Leu Lys Glu Glu Leu Ala Tyr Leu Lys Lys Asn His Glu Glu Glu 245 250 255 Met Asn Ala Leu Arg Gly Gln Val Gly Gly Asp Val Asn Val Glu Met 260 265 270 Asp Ala Ala Pro Gly Val Asp Leu Ser Arg Ile Leu Asn Glu Met Arg 275 280 285 Asp Gln Tyr Glu Lys Met Ala Glu Lys Asn Arg Lys Asp Ala Glu Glu 290 295 300 Trp Phe Phe Thr Lys Thr Glu Glu Leu Asn Arg Glu Val Ala Thr Asn 305 310 315 320 Ser Glu Leu Val Gln Ser Gly Lys Ser Glu Ile Ser Glu Leu Arg Arg 325 330 335 Thr Met Gln Asn Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys 340 345 350 Ala Ser Leu Glu Asn Ser Leu Glu Glu Thr Lys Gly Arg Tyr Cys Met 355 360 365 Gln Leu Ala Gln Ile Gln Glu Met Ile Gly Ser Val Glu Glu Gln Leu 370 375 380 Ala Gln Leu Arg Cys Glu Met Glu Gln Gln Asn Gln Glu Tyr Lys Ile 385 390 395 400 Leu Leu Asp Val Lys Thr Arg Leu Glu Gln Glu Ile Ala Thr Tyr Arg 405 410 415 Arg Leu Leu Glu Gly Glu Asp Ala His Leu Ser Ser Ser Gln Phe Ser 420 425 430 Ser Gly Ser Gln Ser Ser Arg Asp Val Thr Ser Ser Ser Arg Gln Ile 435 440 445 Arg Thr Lys Val Met Asp Val His Asp Gly Lys Val Val Ser Thr His 450 455 460 Glu Gln Val Leu Arg Thr Lys Asn 465 470 <210> 4 <211> 382 <212> PRT <213> Homo sapiens <400> 4 Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp Lys Val Gln Ala 1 5 10 15 Tyr Ser Thr Ala Gly Gly Lys Val Trp Leu Ser Val Leu Phe Ile Phe 20 25 30 Arg Ile Leu Leu Leu Gly Thr Ala Val Glu Ser Ala Trp Gly Asp Glu 35 40 45 Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly Cys Glu Asn Val 50 55 60 Cys Tyr Asp Lys Ser Phe Pro Ile Ser His Val Arg Phe Trp Val Leu 65 70 75 80 Gln Ile Ile Phe Val Ser Val Pro Thr Leu Leu Tyr Leu Ala His Val 85 90 95 Phe Tyr Val Met Arg Lys Glu Glu Lys Leu Asn Lys Lys Glu Glu Glu 100 105 110 Leu Lys Val Ala Gln Thr Asp Gly Val Asn Val Asp Met His Leu Lys 115 120 125 Gln Ile Glu Ile Lys Lys Phe Lys Tyr Gly Ile Glu Glu His Gly Lys 130 135 140 Val Lys Met Arg Gly Gly Leu Leu Arg Thr Tyr Ile Ile Ser Ile Leu 145 150 155 160 Phe Lys Ser Ile Phe Glu Val Ala Phe Leu Leu Ile Gln Trp Tyr Ile 165 170 175 Tyr Gly Phe Ser Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys 180 185 190 Pro His Gln Val Asp Cys Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile 195 200 205 Phe Ile Ile Phe Met Leu Val Val Ser Leu Val Ser Leu Ala Leu Asn 210 215 220 Ile Ile Glu Leu Phe Tyr Val Phe Phe Lys Gly Val Lys Asp Arg Val 225 230 235 240 Lys Gly Lys Ser Asp Pro Tyr His Ala Thr Ser Gly Ala Leu Ser Pro 245 250 255 Ala Lys Asp Cys Gly Ser Gln Lys Tyr Ala Tyr Phe Asn Gly Cys Ser 260 265 270 Ser Pro Thr Ala Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Leu 275 280 285 Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn Tyr Asn Lys Gln 290 295 300 Ala Ser Glu Gln Asn Trp Ala Asn Tyr Ser Ala Glu Gln Asn Arg Met 305 310 315 320 Gly Gln Ala Gly Ser Thr Ile Ser Asn Ser His Ala Gln Pro Phe Asp 325 330 335 Phe Pro Asp Asp Asn Gln Asn Ser Lys Lys Leu Ala Ala Gly His Glu 340 345 350 Leu Gln Pro Leu Ala Ile Val Asp Gln Arg Pro Ser Ser Arg Ala Ser 355 360 365 Ser Arg Ala Ser Ser Arg Pro Arg Pro Asp Asp Leu Glu Ile 370 375 380

Claims (29)

1) contacting a biological sample obtained from a mammal with actin, connexin-43 and keratin antigens, respectively, or antigen fragments thereof to bind the target autoantibody; and
2) detecting the presence of the target autoantibody in the sample by detecting binding of the target autoantibody to the respective antigen;
A method for detecting target autoantibodies to actin, connexin-43 and keratin in a mammal, comprising: The method of claim 1 , wherein the actin comprises alpha myocardial actin (ACTC1), alpha skeletal muscle actin (ACTA1), or a combination thereof. The method of claim 1 , wherein the keratin is keratin-24. The method of claim 1 , wherein actin, keratin and connexin-43 are human proteins. The method of claim 1 , wherein an antigenic fragment of actin comprising a sequence of at least about 10 amino acids from SEQ ID NO: 1 or SEQ ID NO: 2 is used. The method according to claim 1 , wherein an antigenic fragment of keratin comprising a sequence of at least about 10 amino acids from SEQ ID NO:3 is used. The method of claim 1 , wherein an antigenic fragment of connexin-43 comprising a sequence of at least about 10 amino acids from SEQ ID NO:4 is used. The method of claim 1 , wherein the antigen or antigenic fragment is bound to a solid support. The method of claim 1 , wherein the bound target autoantibody is detected using a labeled secondary antibody. The method according to claim 9, wherein the secondary antibody is labeled with an enzyme label, a fluorescent label, an affinity label or a radioactive label. The method of claim 1 , which is an ELISA. The method according to claim 1, which is a western blotting method. The method of claim 1 , wherein the biological sample is a serological sample. 1) contacting a biological sample obtained from a mammal with actin, connexin-43 and keratin antigens, respectively, or antigen fragments thereof, to bind the target autoantibody to the antigen;
2) detecting the presence of the target autoantibody in the sample by detecting binding of the target autoantibody to the respective antigen; and
3) diagnosing the mammal as having Brugada syndrome when the presence of the target autoantibody is detected
A method for diagnosing Brugada syndrome in a mammal, comprising: The method of claim 14 , wherein the actin comprises alpha myocardial actin (ACTC1), alpha skeletal muscle actin (ACTA1), or a combination thereof. 15. The method of claim 14, wherein the keratin is keratin-24. 15. The method of claim 14, wherein actin, keratin and connexin-43 are human proteins. 15. The method of claim 14, wherein an antigenic fragment of actin comprising a sequence of at least about 10 amino acids from SEQ ID NO: 1 or SEQ ID NO: 2 is used. 15. The method according to claim 14, wherein an antigenic fragment of keratin comprising a sequence of at least about 10 amino acids from SEQ ID NO:3 is used. 15. The method of claim 14, wherein an antigenic fragment of connexin-43 comprising a sequence of at least about 10 amino acids from SEQ ID NO:4 is used. 15. The method of claim 14, wherein the antigen or antigenic fragment is bound to a solid support. The method of claim 14 , wherein the bound target autoantibody is detected using a labeled secondary antibody. 23. The method of claim 22, wherein the secondary antibody is labeled with an enzyme label, a fluorescent label, an affinity label or a radioactive label. 15. The method of claim 14, which is an ELISA. 15. The method of claim 14, which is a Western blotting method. The method of claim 14 , wherein the biological sample is a serological sample. 1) contacting a biological sample obtained from a mammal with actin, connexin-43 and keratin antigens, respectively, or antigen fragments thereof to bind the target autoantibody;
2) detecting the presence of the target autoantibody in the sample by detecting binding of the target autoantibody to the respective antigen;
3) diagnosing the mammal as having Brugada syndrome when the presence of the target autoantibody is detected; and
4) treating the mammal with one or more of an implantable defibrillator, catheter ablation, and a drug.
A method for diagnosing and treating Brugada syndrome in a mammal, comprising: 28. The method of claim 27, wherein the drug is selected from therapies that rebalance the current active during phase 1 of the right ventricular action potential to stop the electrical storm and increase the calcium channel current. 28. The method of claim 27, wherein the drug is selected from class IA antiarrhythmic agents, β-adrenergic agonists and phosphodiesterase III inhibitors.

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