Use of reagents for detecting mutations in the SCN2B and / or SCN4B genes in the manufacture of a product for detecting genetic arrhythmias

Abstract

The application discloses application of a reagent for detecting SCN2B and / or SCN4B gene mutation in preparation of a genetic arrhythmia detection product. Through a double detection system of whole exon sequencing combined with Sanger sequencing verification, the application realizes accurate capture of pathogenic mutations such as SCN2B-R28Q / Y69H / P210L and SCN4B-T211M, and solves the difficulty that only SCN5A is confirmed as a main pathogenic gene in the current clinical diagnosis of J wave syndrome (JWS), and about 70%-80% of patients have gene detection negative. The three missense mutations of SCN2B and the SCN4B-T211M variation are reported for the first time in JWS, and SCN4B is confirmed as a pathogenic gene of JWS for the first time, which significantly expands the pathogenic gene spectrum of JWS. The detection product developed based on this can realize early accurate diagnosis, provide personalized risk assessment (such as early warning of sudden death induced by fever) for patients, and guide genetic screening of family members, and significantly improves the clinical diagnosis and treatment efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of reagents for detecting SCN2B and / or SCN4B gene mutations in the preparation of products for detecting hereditary arrhythmias. Background Technology

[0002] Hereditary arrhythmias refer to a group of heart diseases caused by gene mutations and that can be passed down within a family. Under normal circumstances, the heart's electrical activity proceeds in an orderly manner, with each component working in close coordination to ensure regular contraction and relaxation. However, in patients with hereditary arrhythmias, gene mutations lead to disordered electrical activity in the heart. Once the heart's electrical activity becomes abnormal, it significantly increases the risk of serious consequences such as sudden death and heart failure. J-wave syndromes (JWS) include early repolarization syndrome (ERS) and Brugada syndrome (BrS), characterized by prominent J waves accompanied by ST-segment elevation on the electrocardiogram (ECG). These syndromes are prone to causing polymorphic ventricular tachycardia and ventricular fibrillation, leading to sudden cardiac death. Brugada syndrome is characterized by arched ST-segment elevation in the right precordial leads, followed by negative T waves. Early repolarization ECG is characterized by a prominent J wave or J-point elevation, notching, or stuttering at the end of the QRS complex. Mutations in the SCN5A gene have been found in 20-25% of BrS patients. Other rare variants involving more than 20 genes encoding proteins related to Nav1.5 regulation (INa), L-type calcium channels (ICa), or potassium channel subunits (Ito and IK, ATP) have also been reported as susceptibility factors for BrS. SCN5A variants account for 10% of ERS cases. In addition to SCN5A, nine other genes have been confirmed to be associated with ERS.

[0003] Voltage-gated sodium channels (VGSCs) play a crucial role in the functional realization of excitatory tissues, a fact widely recognized. The coordinated electrical activity of living cells in excitatory tissues relies on the participation of VGSCs and their helper subunits. In cardiomyocytes, the predominantly expressed α subunit is Nav1.5, encoded by the human SCN5A gene. To date, hundreds of SCN5A gene mutations have been found to be associated with hereditary arrhythmias, involving a diverse range of diseases, including long QT syndrome, Brugada syndrome (BrS), early repolarization syndrome (ERS), cardiac conduction defects, atrial fibrillation, idiopathic ventricular fibrillation, and sudden infant death syndrome. Notably, a single SCN5A mutation can simultaneously trigger multiple phenotypes, forming overlap syndromes. This important finding fully highlights the critical role of Nav1.5 in maintaining physiological states and in the development and progression of diseases.

[0004] Nav1.5 does not function independently; it interacts with and is regulated by various proteins to form a large sodium complex. These interacting proteins include the β subunit, Kir2.1, Nav1.8, α1-mutatrophic protein, caveolin-3, glycerol-3-phosphate dehydrogenase-1-like protein, myelin oligodendrocyte glycoprotein 1, desmosome plaque-like protein 2, fibroblast growth factor homolog, and synapse-associated protein 97. Among these interacting proteins, the role of the β subunit in the development of hereditary arrhythmias is receiving increasing attention. There are five β subunits of the VGSC: β1, β1b, β2, β3, and β4. All of them possess an extracellular immunoglobulin domain, which is homologous to the V-set superfamily of cell adhesion molecules. Structurally, β1, β2, β3, and β4 are single transmembrane proteins exhibiting a type I topology, consisting of an extracellular N-terminus, a single transmembrane segment, and an intracellular C-terminus. β1b, however, differs in that it retains its third intron through alternative splicing, ultimately forming a soluble intracellular subtype lacking a transmembrane domain. All β subunits are expressed in cardiac tissue and play a role in several key processes associated with Nav1.5. Specifically, they participate in Nav1.5 transport, influencing its biogenesis and regulating its gating function; they also recruit cytoskeletal aptamers, enzymes, and signaling molecules, and play a crucial mediating role in the adhesion of Nav1.5 to the cytoskeletal framework and extracellular matrix. Current research indicates that all β subunits are associated with arrhythmia phenotypes, encompassing a wide range of inherited arrhythmias, including long QT syndrome, BrS, ERS, cardiac conduction defects, atrial fibrillation, idiopathic ventricular fibrillation, and sudden infant death syndrome.

[0005] Research into the intrinsic link between the β subunit and cardiac arrhythmias provides new directions for our in-depth understanding of the pathogenesis of cardiac arrhythmias. By further exploring the mechanism of action of the β subunit, we hope to develop novel treatments targeting the β subunit, bringing new hope to patients with cardiac arrhythmias. Summary of the Invention

[0006] The main objective of this invention is to propose the application of reagents for detecting SCN2B and / or SCN4B gene mutations in the preparation of products for detecting hereditary arrhythmias. The aim is to explain the mechanism by which SCN2B and SCN4B gene mutations lead to JWS through action potential and spatial structure models, and to construct cell models for the diagnosis and treatment of SCN2B and SCN4B gene mutations.

[0007] To achieve the above objectives, this invention proposes the application of reagents for detecting SCN2B and / or SCN4B gene mutations in the preparation of products for detecting hereditary arrhythmias.

[0008] Preferably, the whole exon sequence of the SCN2B gene is shown in SEQ ID NO. 1, and the whole exon sequence of the SCN4B gene is shown in SEQ ID NO. 2.

[0009] Preferably, the reagent is used to detect R28Q, Y69H, P210L mutations in the SCN2B gene and / or T211M mutations in the SCN4B gene.

[0010] Preferably, the reagent includes probes for detecting SCN2B and / or SCN4B gene mutations.

[0011] Preferably, the probe is capable of specifically recognizing the R28Q, Y69H, and P210L mutations in the SCN2B gene and / or the T211M mutation in the SCN4B gene.

[0012] Preferably, the detection product includes a preparation, a gene chip, or a reagent kit.

[0013] Preferably, the detection product is a PCR kit, which contains primers for amplifying SCN2B and / or SCN4B gene fragments, DNA polymerase, dNTPs, and PCR reaction buffer.

[0014] Preferably, the test sample for the test product is peripheral blood, oral swab, or myocardial biopsy tissue.

[0015] Preferably, the detection product is used for early diagnosis, risk assessment, and prognosis of hereditary arrhythmias.

[0016] Preferably, the testing product is used to screen for personalized treatment plans suitable for patients with hereditary arrhythmias.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) This invention utilizes a dual detection system validated by whole-exome sequencing and Sanger sequencing to achieve precise detection of pathogenic mutations such as SCN2B-R28Q / Y69H / P210L and SCN4B-T211M. This overcomes the current predicament in clinical diagnosis of J-wave syndrome (JWS) where only SCN5A is identified as the main pathogenic gene, and approximately 70%-80% of patients have negative gene tests. Among these, the three missense mutations of SCN2B and the SCN4B-T211M variant are reported for the first time in JWS, and SCN4B is identified as a pathogenic gene for JWS for the first time, significantly expanding the pathogenic gene spectrum of JWS. The detection product developed based on this can achieve early and accurate diagnosis, provide patients with personalized risk assessment (such as early warning of sudden death induced by fever), and guide genetic screening of family members, significantly improving the efficiency of clinical diagnosis and treatment.

[0019] (2) This invention, through a multi-dimensional technical system of whole-cell patch-clamp, confocal microscopy, structural modeling, and action potential simulation, systematically elucidates for the first time the dual pathogenic mechanism of SCN2B and SCN4B mutations: On the one hand, SCN2B and SCN4B mutations cause Nav1.5 structural instability by breaking or forming local spatial hydrogen bonds, which, combined with channel transport barriers observed by confocal microscopy, jointly trigger I Na The SCN4B-T211M mutation reduces the transient outward potassium current (Ik) through its interaction with Kv4.3. to The increase in sodium-potassium current imbalance is the core electrophysiological basis for JWS. Based on this, a human ventricular cell action potential model was developed, further revealing the pathological process by which mutations under febrile conditions can induce an increase in the transventricular repolarization voltage gradient, providing theoretical support for clinical avoidance of triggering factors. Simultaneously, the constructed TSA201 mutant stable cell line (capable of testing 500 compounds daily) and gene knock-in animal model form a complete translational platform from mechanism research to high-throughput drug screening and efficacy evaluation. This platform can provide a basis for targeted drug design for key sites predicted by the AlphaFold structural model, and accelerate the development of novel antiarrhythmic drugs, achieving a closed-loop diagnosis and treatment system from etiological diagnosis to mechanism analysis and precision treatment. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a clinical and genetic analysis diagram of SCN2B gene mutation JWS carriers in this invention; A: SCN2B-R28Q family pedigree; B and C: ECGs of the SCN2B-R28Q proband and his mother after ajmaline provocation test; F and G: ECGs of two unrelated SCN2B-Y69H probands after flecain and ajmaline provocation tests; J, K, and N: Standard ECG (J) and flecain provocation ECG (K) of a proband carrying SCN2B-P210L, with a history of atrial fibrillation and early repolarization (N); D, H, and L: Diagrams of SCN2B-R28Q heterozygous mutations in patients; E, I, and M: Comparison of exon sequencing data between normal and mutation-carrying patients.

[0022] Figure 2The diagram shows the clinical and genetic analysis of JWS carriers of the SCN4B gene mutation in this invention; A and B: Electrocardiograms of the proband carrying SCN4B-T211M during fever and after the flecainide challenge test; C: Comparison of exon sequencing data between normal and mutation-carrying patients; D: Comparison of the conservation of SCN4B-T211M in different mammalian species; E: Location of SCN2B and SCN4B mutation sites in the conventional transmembrane topology model of SCN5A.

[0023] Figure 3 This diagram illustrates the electrophysiological effects of co-expression of SCN2B mutation and SCN5A on cardiac sodium channels INa in TSA201 cells. A and C: Representative records and IV relationship diagrams of peak INa values ​​when co-expressed with SCN5A-WT+SCN2B-WT, or with SCN2B-R28Q, Y69H, and P210L mutants. B and D: Representative records and steady-state inactivation curves (n=8-14) of voltage-dependent inactivation of the channel when co-expressed with SCN5A-WT+SCN2B-WT, or with SCN2B-R28Q, Y69H, and P210L mutants. E: Confocal fluorescence microscopy images of GFP-labeled SCN5A channel protein co-expressed with SCN2B / WT or mutants. The upper and lower micrographs show phase-contrast transmission images and single-fluorescence confocal images of the same cell center, respectively. F: Images of SCN5A-WT co-transfected with SCN2B-P210L. Left: Transmission micrograph of target cells (ad). Middle: Red signals represent cells successfully transfected with SCN2B using the pIRES2-DsRed-Express vector. Right: Green signals represent the cell distribution of GFP-labeled SCN5A channel protein.

[0024] Figure 4 The diagram shows the electrophysiological effects of the SCN4B mutation (SCN4B-T211M) of this invention on cardiac INa when co-expressed with SCN5A in TSA201 cells; A and B: Representative peak INa records and IV relationship diagrams of SCN4B-WT or SCN4B-T211M mutants at different temperatures; C: Voltage-dependent channel inactivation and activation curves of SCN4B-WT or SCN4B-T211M mutants at different temperatures; D: Relationship between total charge and voltage of INa current in the first 50 ms at different temperatures; E and F: Representative records of rapid inactivation recovery of SCN4B-WT or SCN4B-T211M mutants at different temperatures and comparison of rapid inactivation recovery time constants, measured using the dual-pulse protocol shown in the inset.

[0025] Figure 5The following is a diagram showing the effect of SCN4B-T211M on transient outward potassium current (Ito) in this invention; A: Representative Ito current traces recorded in cells expressing KCND3-WT and co-transfected with SCN4B-WT or SCN4B-T211M, with the inset showing the voltage clamping protocol used; B: Current-voltage relationship of peak Ito recorded in cells co-expressing KCND3-WT and SCN4B-WT or SCN4B-T211M.

[0026] Figure 6 The diagram shows the simulated human ventricular action potentials induced by SCN4B-WT and mutations (considering the effects of fever and increased Ito at different pacing cycle lengths); Ai, Bi, Ci: Action potentials of epicardial, medullary, and endocardial cells under wild-type conditions at a basic cycle length (BCL) of 1000 ms (green solid line), BCL=400 ms (red solid line), wild-type fever (brown solid line), and wild-type fever with different fold increases in Ito channel conductance (orange dashed line: double increase; blue dashed line: 100-fold increase, all at BCL=400 ms); Ai, Bi, Ci: Action potentials of epicardial, medullary, and endocardial cells under mutant conditions at BCL=1000 ms (green solid line), BCL=400 ms (red solid line), mutant fever (brown solid line), and mutant fever with different fold increases in Ito channel conductance (orange dashed line: double increase; blue dashed line: 100-fold increase, all at BCL=400 ms). Action potential (ms).

[0027] Figure 7 The diagram shows the protein-protein interaction and structural analysis of SCN2B and SCN4B in this invention; A and B: gene relationship maps of SCN2B and SCN4B based on STRING network analysis; C, D, E, and F: diagrams predicting changes in spatial structure of SCN2B due to mutations based on AlphaFold structural modeling.

[0028] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0031] Example 1: Verification of gene mutations using whole-exome sequencing and Sanger sequencing

[0032] 1. Sample collection

[0033] This invention selected 269 patients diagnosed with gene-negative JWS between January 2020 and December 2023. The diagnosis of JWS was confirmed by electrocardiogram and medical history inquiry. All patients were fully informed, informed of the purpose of the procedure, and obtained their consent. After signing the informed consent form, 5 mL of peripheral venous blood was collected. At the same time, peripheral blood or oral swabs from family members (parents and children) (2 cases) were collected as controls for DNA extraction.

[0034] 2. DNA extraction

[0035] DNA extraction: A commercial kit (such as the QIAamp DNA Blood Mini Kit) was used, and the procedure was followed according to the instructions. Peripheral blood samples were lysed with red blood cells and white blood cells to precipitate DNA. Oral swabs / myocardial tissue samples were digested with proteinase K to extract DNA.

[0036] Quality control: DNA concentration and purity were determined using Nanodrop 2000, requiring a 260 / 280 ratio >1.8. DNA integrity was verified by 1% agarose gel electrophoresis (no obvious degradation bands). Qualified DNA was stored at -20℃ for later use.

[0037] 2. Whole exome sequencing and mutation annotation

[0038] Genomic DNA was randomly fragmented into 150-300 bp main band fragments using an ultrasonic fragmentation device (Covaris M220, Massachusetts, USA). 500 ng of purified DNA fragments were used for end repair and ligation of marker adapters to construct DNA sequencing libraries. A three-step enzymatic reaction was performed according to the Illumina standard library construction method: end repair, addition of "A" markers, and ligation of Illumina sequencing adapters (an 8 bp barcode was ligated to the DNA fragment via PCR) to form the sample library. Exon capture was performed using Agilent SureSelect; see the instruction manual for details.

[0039] Sequencing was performed using the Illumina HiSeq X-ten platform. SNP sites and insertion / deletion markers were detected using GATK v3.5, and annotation was performed using ANNOVAR.

[0040] Bioinformatics prediction tools were used to predict the conservation and pathogenicity of candidate variants. All variants were compared with the public databases 1000 Genomes Project (http: / / www.internationalgenome.org / ), Exomevariant server, NHLBI GO Exome Sequencing Project (ESP) (http: / / evs.gs.washington.edu / EVS / ), and Exome Aggregation Consortium (ExAC) (http: / / exac.broadinstitute.org / ) to screen for pathogenic variants.

[0041] 3. Sanger sequencing verification

[0042] The mutation sites were validated using Sanger sequencing on an ABI 3730 sequencer. The SCN2B and SCN4B mutations were detected by comparison with reference sequences (NCBI: SCN2B:NM_004588.5; SCN4B NM_174934.4). The whole exon sequences of the SCN2B and SCN4B genes are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0043] 4. Results

[0044] The results are as follows Figure 1 and Figure 2 As shown, Figure 1A is the pedigree of Patient 1, a 32-year-old asymptomatic male who tested positive for ajmaline provocation. His mother also tested positive for ajmaline provocation. Figure 1 B, 1C). The SCN2B-R28Q heterozygous mutation was detected in both of them. Figure 1 D).

[0045] Patient 2 was a 48-year-old asymptomatic male. His grandmother died suddenly at the age of 26, and two of his sisters had experienced miscarriages. A flecainide provocation test induced convex ST-segment elevation in the right precordial leads. Figure 1 F). Patient 3 was a 42-year-old male presenting with recurrent syncope, with no positive family history. An ajmaline provocation test revealed a typical Brugada type 1 electrocardiogram (F). Figure 1 G). Patient 3's electrocardiogram showed first-degree atrioventricular block. A heterozygous missense SCN2B-Y69H mutation with identical exon 2 of SCN2B was found in both patients 2 and 3. Figure 1 H).

[0046] Patient 4 was a 41-year-old asymptomatic male whose electrocardiogram spontaneously showed Brugada type 1 and global early repolarization patterns. Figure 1 J), and a history of recurrent atrial fibrillation ( Figure 1 N), previously treated with propafenone for cardioversion. The flecainide provocation test ECG confirmed the typical Brugada pattern in leads V1-V3, while the early repolarization pattern in other leads was reduced as expected. Figure 1 K). This case revealed a heterozygous missense mutation SCN2B-P210L in exon 4 of SCN2B (K). Figure 1 L).

[0047] Patient 5 was a 46-year-old asymptomatic male with a family history of sudden cardiac death. He presented with a typical Brugada type 1 electrocardiogram during fever. Figure 2 A), the flecainide challenge test further confirmed ( Figure 2 B). A rapid episode of ventricular tachycardia was recorded during electrophysiological examination, followed by ICD implantation. This case revealed a novel missense mutation in SCN4B (base position 635 changed from C to T), resulting in the change of amino acid position 211 from threonine (T) to methionine (M). Figure 2 C). These variations were not found in more than 800 controls and are highly conserved across different species. Figure 1 D, 1I, 1M, and 2D). SCN2B-R28Q and SCN2B-Y69H are located in the extracellular domain; SCN2B-P210L and SCN4B-T211M are located in the intracellular domain. Figure 2 E).

[0048] Example 2 Functional verification of SCN2B and SCN4B mutations

[0049] 1. Cell model construction

[0050] (1) Plasmid construction

[0051] Wild-type plasmids: The sequences of SCN2B-WT, SCN4B-WT and SCN5A-WT (Nav1.5 encoding gene) were amplified from the human cDNA library, cloned into the pIRES2-DsRed-Express vector (containing red fluorescent label), and the correctness of the inserted sequence was verified by enzyme digestion (XhoI / BamHI) and sequencing.

[0052] Mutant plasmids: SCN2B-R28Q / Y69H / P210L and SCN4B-T211M mutant plasmids were constructed using the QuikChange site-directed mutagenesis kit (Agilent, USA). The mutation sites were confirmed by sequencing.

[0053] Control plasmid: pcDNA3.1 empty vector (containing GFP fluorescent label), used for transfection efficiency assessment.

[0054] (2) Cell culture and transfection

[0055] Cell line: TSA201 cells (human embryonic kidney cells, without endogenous Nav1.5 expression) were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco, USA), 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator.

[0056] Transfection method: FuGENE®6 transfection reagent (Promega, USA) was used. The ratio of FuGENE®6 transfection reagent to mixed plasmids (SCN2B-WT / R28Q, SCN2B-WT / Y69H, SCN2B-WT / P210L, SCN4B-WT / T211M +SCN5A-WT, 1:1) was 1:1. FuGENE®6 was added, and the mixture was incubated at room temperature for 15 minutes. TSA201 cells were then added to 6-well plates (cell density 5 × 10⁶). 5 / well), and simultaneously transfect GFP control plasmid (0.5 μg / well). After 48 hours, observe fluorescence (red + green) to assess transfection efficiency (≥60%).

[0057] 2. Electrophysiological analysis (patch clamp)

[0058] (1) I Na Record

[0059] Solution preparation:

[0060] I Na(Sodium current) Recording internal solution (mmol / L): 5 NaCl, 5 KCl, 130 CsF, 1.0 MgCl2, 5 EGTA, 10 HEPES, pH adjusted to 7.2 with CsOH;

[0061] I Na Record the external solution (mmol / L): 130 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 2.8 NaAc, 10 HEPES, 10 Glucose, and adjust the pH to 7.3 with NaOH.

[0062] Current density curve (IV curve) and voltage-dependent steady-state activation:

[0063] (1) I Na The current density curve (IV curve) and the voltage-dependent steady-state activation stimulation scheme are as follows: the holding potential (HP) is -120mV, the command potential starts from -90mV or -80mV and jumps to +30 or +60mV in 5mV steps, the pulse width is 30ms, and the sampling frequency is 20kHz.

[0064] (2) A downward-directed, rapidly activated and rapidly deactivated current can be recorded, which can be blocked by tetrodotoxin, i.e., I Na .

[0065] (3) Read the data and plot the voltage-current relationship curve. The steady-state activation curve is the calculated conductance (G) obtained after converting the IV curve into membrane conductivity: G = I peak / (V test -V res In this formula, I peak For peak current, V test V represents the command potential, and Vres represents the reversal potential (the membrane potential when the current is 0). V... test G / G is the x-axis. max Using the ordinate as the vertical axis, according to the Boltzmann equation: G(v) / G max =1 / {1+exp[(V m -V 1 / 2 ) / k-1}, to fit and plot the voltage-dependent steady-state activation curve. In the formula, G represents the voltage-dependent steady-state activation curve of the whole-cell Nav1.5 channel at each V. m The conductivity under the condition, G max This represents the maximum conductivity of the whole-cell Nav1.5 channel, V. m V1 / 2 represents the membrane potential, V1 / 2 represents the half-activation voltage, i.e., the voltage value when half of the channels are activated, and k is the slope factor.

[0066] Steady-state deactivation curve:

[0067] (1) Record I Na The steady-state inactivation curve was obtained using the double-pulse stimulation method with a power of -120mV. The conditional pulse started at -140mV, depolarized to -60mV every 5mV for 100ms, and was followed by a test pulse depolarized to -20mV for 30ms.

[0068] (2) When plotting the steady-state deactivation curve, the conditional pulse is used as the abscissa, and the peak value of the Nav1.5 current in the test pulse (I) is used as the ordinate. max The denominator is the Nav1.5 current value (I) obtained for each test pulse, and the numerator is the Nav1.5 current value (I) obtained for each test pulse. Calculate the ratio of the current to the peak current for each test pulse (I / I0). max ), and use this as the ordinate to plot the steady-state deactivation curve.

[0069] (3) Similarly, according to the Boltzmann equation I(v) / I max =l / {l+exp[(V m -V 1 / 2 ) / K]}, to fit the steady-state deactivation curve. Where I is the value of each V m Peak current under, I max The maximum peak current is V. t V 1 / 2 is the half-deactivation voltage, i.e., the voltage value when half of the channels are deactivated, and k is the slope factor.

[0070] (4) Record I Na The steady-state inactivation recovery curve was obtained using a double-pulse stimulation method. The HP was -120 mV, and two identical stimuli (P1 and P2) were applied at -20 mV for 30 ms, with the interval between the two stimuli maintained at HP-120 mV. The interval duration Δt increased sequentially: 0.1, 0.3, 0.5, 1, 2, 3, 5, 8, 12, 20, 50, 100, 300, 500, and 1000 ms, with a sampling frequency of 20 kHz. The peak currents corresponding to P1 and P2 were recorded, and their ratios were calculated. The inactivation recovery time constant was fitted using a double exponential method, I(t) / I0. max =Af·(1-exp(-t / τf))+As·(1-exp(-t / τs)), where τf and τs are the fast activation time constant and the slow activation time constant, respectively, and Af and As correspond to the amplitudes of the fast and slow phases.

[0071] Experimental results:

[0072] Depend on Figure 3 and 4 It can be known that: Figure 3 A shows the macroscopic current records for these channels. Figure 3C shows the corresponding IV relationship curves, with the maximum peak inward current of all channels occurring at -35 mV. When SCN2B was co-expressed with SCN5A-WT, the peak INa current density decreased from -554.3 ± 50.5 pA / pF (SCN2B-WT) to -382.3 ± 47.2 pA / pF (SCN2B-R28Q), -321.6 ± 36.1 pA / pF (SCN2B-Y69H), and -157.7 ± 34.1 pA / pF (SCN2B-P210L) (p < 0.05, 0.01, 0.01 compared to the SCN2B-WT group). Notably, we observed that 35.7% of cells co-expressing SCN2B-P210L exhibited complete loss of function. Figure 3 B). Steady-state activation and inactivation were similar across the three mutant groups. Figure 3 C and 3D). Inactivation recovery measured by the standard double-pulse protocol showed that there was a delayed recovery in all three mutant groups compared with the WT group, and the differences were statistically significant (p<0.05, 0.05, 0.01, respectively, compared with the WT group).

[0073] To investigate whether the loss of function caused by the SCN2B mutation is partly due to a transport defect, we co-expressed GFP fusions with SCN5A-WT and SCN2B-WT or mutants (with RFP) in a bicistronic vector. XYZ scanning of SCN5A-WT+SCN2B-WT under a confocal microscope revealed a central and peripheral staining pattern, indicating that sodium channels are simultaneously localized in the cell membrane and intracellular organs. Figure 3 E. Leftmost panel). SCN2B-R28Q and SCN2B-Y69H have virtually no impact on the transfer of Nav1.5 ( Figure 3 E. Second and third left-hand panels). In contrast, in some SCN2B-P210L cells, staining was confined to the cell interior, exhibiting characteristics of internalization, and no staining was observed on the cell membrane, suggesting that sodium channels may be trapped in the endoplasmic reticulum and / or Golgi complex (E). Figure 3 (E, fourth left panel). Meanwhile, some Nav1.5 protein can still be transported to the cell membrane, consistent with electrophysiological results. Figure 3 (E, fifth left panel). For further comparison... Figure 3F shows that when cells did not take up the carrier containing SCN2B-P210L (cell b), the Nav1.5 channel showed obvious staining and normal transport to the cell membrane; while when SCN2B-P210L was highly expressed in the target cells (cell d), typical transport defects were observed; for cells with moderate SCN2B-P210L expression levels (cells a and c), the transport level of Nav1.5 was between the two. The ratio of peripheral to total cell area fluorescence intensity was similar in the following three groups: SCN5A-WT+SCN2B-WT (69.39±8.72%), SCN5A-WT+SCN2B-R28Q (60.52±10.18%), and SCN5A-WT+SCN2B-Y69H (7.47±11.65%) (p>0.05 for SCN2B-R28Q or Y69H compared to WT). However, it decreased significantly in the SCN5A-WT+SCN2B-P210L group (23.59±14.27%; p<0.05 compared with WT), indicating that SCN2B-P210L significantly impaired the translocation capacity of the Nav1.5 channel.

[0074] Figure 4 At 22°C, the peak INa density was comparable between the SCN4B-WT group and the mutant group. However, at 34°C, co-expression of SCN4B-T211M and SCN5A-WT significantly reduced the peak current density compared to SCN4B-WT+SCN5A-WT (-570.01 ± 34.46 pA / pF vs. -431.6 ± 25.68 pA / pF, P < 0.01). Figure 4 A-4B). At room temperature (22°C), steady-state activation, inactivation, and inactivation recovery showed no significant differences between WT and T211M. However, at 34°C, SCN4B-T211M significantly shifted steady-state activation to a more negative potential (-64.41 ± 1.90 mV vs. -55.88 ± 1.20 mV, n=6, 6; P<0.05). Figure 4 C), and delayed Tf (5.49 ± 1.10 ms vs. 1.28 ± 0.14 ms, n=6, 6; P<0.05, Figure 4 E and Figure 4 F), compared with SCN4B-WT+SCN5A-WT. Although the total sodium charge in both groups increased with increasing temperature, the SCN4B-T211M mutation significantly reduced the sodium charge at 34°C: a reduction of 61.17% at -20 mV (-4250.57 ± 902.79 pC vs. -1650.56 ± 193.84 pC, n=6, 6; P<0.05, Figure 4D), decreased by 54.91% at -10 mV (-3341.45 ± 718.74 pC vs. -1506.56 ± 194.11 pC, n=6, 6; P<0.05, Figure 4 D). These effects were not evident in the SCN4B-T211M group at 22°C. Within the range of -20mV to 0mV, the open-state rapid and chronic decay time constants of WT and T211M were similar at 22°C, while at 34°C, the SCN4B-T211M variant, compared to SCN4B-WT, significantly accelerated the open-state rapid (0.12 ± 0.01 vs. 0.18 ± 0.01, P<0.01) and chronic (0.33 ± 0.11 vs. 0.85 ± 0.18, P<0.05) decay time constants.

[0075] (2) I to Records and Analysis

[0076] Solution preparation:

[0077] I to (Transient outward potassium current) Record the internal solution (mmol / L): 125 KCl, 25 KOH, 1 CaCl2, 2 MgCl2, 4 K-ATP, 10 EGTA, 10 HEPES, and adjust the pH to 7.2 with KOH;

[0078] I to Record the external solution (mmol / L): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 10 Glucose, and adjust the pH to 7.4 with NaOH.

[0079] I to IV curves and voltage-dependent steady-state activation curves: The Ito recording protocol involves first adding CdCl2 (100 mmol / L) to the extracellular fluid to block the L-type calcium current and calcium-activated chloride current, then adding BaCl2 (100 mmol / L) to block the inward rectifying potassium current, and finally adding tetrodotoxin (50 mmol / L) to block I... Na To minimize the influence of the tetrodotoxin-insensitive inward current on the recorded current, a pre-applied pH of -80 mV was used, with CP ranging from -80 mV or -50 mV to +60 mV in increments of 5 mV or 10 mV, and a clamping time of 300 ms. A very distinct and rapid outward current was recorded, activated from -30 mV and increasing with increasing membrane depolarization. This outward current could be blocked by 2 mmol / L 4-AP, indicating that the outward current recorded in this experiment was Ip. to And record the I after administration of the medication (quinidine, cilostazol).to Current changes. To eliminate the error caused by the different sizes of randomly selected cells in the experiment, the current magnitude is expressed as current density (pA / pF). Data is collected and a voltage-current relationship curve is plotted. The steady-state activation curve is obtained by converting the IV curve into membrane electrical properties, and G is calculated as: G = I peak / (V test -V res In this formula, I peak For peak current, V test V represents the command level. res This represents the reversal potential (the membrane potential when the current is 0). In V... test G / G is the x-axis. max Using the ordinate as the vertical axis, according to the Boltzmann equation: G(v) / G max =1 / {1+exp[(V m -V 1 / 2 ) / k-1}, to fit and plot the voltage-dependent steady-state activation curve. In the formula, G represents the whole-cell I to Channels in each V m The conductivity under the condition, G max Indicates whole cell I to Maximum conductance of the channel, V m V represents the membrane potential. 1 / 2 This represents the half-activation voltage, i.e., the voltage value when half of the channels are activated, and k is the slope factor.

[0080] Experimental results:

[0081] Depend on Figure 5 It can be seen that the co-expression of SCN4B-T211M and KCND3-WT significantly enhanced the peak Ito density in the voltage range of -20mV to +40mV. For example, at +40mV, SCN4B-T211M increased the Ito amplitude by 129.8% compared to SCN4B-WT (597.7 ± 22.8 pA / pF vs. 260.1 ± 25.3 pA / pF, P < 0.01). Figure 5 A and Figure 5 B).

[0082] (3) Nav1.5 confocal positioning

[0083] Subcellular localization and transport defects were detected using confocal microscopy. TSA201 cells were seeded in polylysine-coated 35 mm glass-bottomed culture dishes and analyzed 48 hours post-transfection. Imaging was performed using an Olympus FluoView laser scanning confocal microscope (Olympus, Orangeburg, New York, USA), and images were acquired on a personal computer using FluoView acquisition software. GFP-labeled cells were analyzed in an XYZ three-dimensional mode. Excitation light was provided by an argon-ion laser at a wavelength of 488 nm, and emitted light was collected at 520 nm using a photomultiplier tube (PMT) #1; transmission images were acquired at PMT #2. Fluorescence signals were collected using 40× or 60× oil immersion objectives. The XY frame was set to 512×512 pixels, and the laser intensity was adjusted to 6%. The Z-axis was scanned across the entire cell volume in approximately 0.50 μm steps, controlled by a computer. To quantitatively analyze the fluorescence intensity of Nav1.5 in the plasma membrane region (2 μm range) and the entire cell area, measurements were taken in the intermediate XY images of Z-series stacks, and the ratio of membrane region fluorescence intensity to total fluorescence intensity was calculated. Analysis of GFP-labeled cells was performed using Fluoview and ImageJ software.

[0084] Example 3: Action Potential and Spatial Structure Model Explains the JWS Mechanism Caused by Mutations

[0085] This study employed the Human Ventricular Cell Action Potential (TNNP) model developed by ten Tusscher et al. This model was chosen based on experimental data from human ventricular myocytes and has been validated, while also considering the transmural heterogeneity of ventricular myocytes in the endocardial, medial, and epicardial regions. The TNNP model has been proven effective in studying the functional effects of gene mutations on the occurrence of cardiac arrhythmias.

[0086] To characterize the impact of the mutation on the ventricular action potential, the TNNP model was modified in the simulation by incorporating experimental data on changes in INa gating characteristics caused by the mutation. These changes included shifts in the activation and inactivation curves (i.e., changes in V1 / 2) and alterations in the channel activation and inactivation time constants. To simulate the effect of fever, the temperature dependence of the channel conductance curves, as described by Abdelsayed et al., was used to adjust the channel conductance from 37°C to 41°C.

[0087] In the simulation, action potentials in the TNNP model were generated by a series of stimuli above the threshold, with the BCL varying from 400 to 1000 milliseconds. The duration of the action potential at 90% repolarization (APD90) was calculated in each case.

[0088] The structures of SCN2B and SCN4B proteins have been fully resolved in the AlphaFold protein structure database, and their full-length amino acid sequences have also been published (https: / / alphafold.com / ). The effects of mutations on protein structure and amino acid interactions were predicted and visualized using Chimera 1.17.3.

[0089] Experimental results: Figure 6 It was found that under WT conditions, as the BCL decreased from 1000 ms to 400 ms, the simulated action potentials of endocardial, medial, and epicardial cells exhibited shortened APDs and lower plateau membrane potentials. However, elevated body temperature (fever) had minimal effect on the action potentials of these three cell types at all considered BCLs, including at BCL = 400 ms. Figure 6 Ai, 6Bi, 6Ci). Mutations further shortened the action potential duration of endocardial, medial, and epicardial cells. At BCL = 1000 ms, the action potential duration (APD) of endocardial, medial, and epicardial cells decreased from 307 ms, 417 ms, and 305 ms under WT conditions to 280 ms, 388 ms, and 282 ms under mutant conditions, respectively. Further reduction of BCL further shortened the action potential of all three cell types. Similar to WT conditions, elevated body temperature had no significant effect on simulated action potentials and APD. Figure 6 Aii, 6Bii, 6Cii).

[0090] It is noteworthy that significant differences were observed between WT and mutant conditions when an increase in transient outward potassium channel maximum conductance (gto) was combined with increased heart rate (i.e., shortened BCL) and increased body temperature. Under WT conditions, a slight increase in gto to two-fold had almost no effect on the action potential, but a significant increase (up to 100-fold) completely eliminated the plateau phase of the action potential, resulting in an "all-or-none" phenomenon in all three cell types, i.e., inability to elicit a response. Under mutant conditions combined with fever, a slight increase in gto also had little effect, but a significant increase led to a marked shortening of the response time in outer and middle membrane cells, with low overshoot amplitude and short response duration, but this phenomenon was not observed in inner membrane cells. Figure 6 (Cii). This result suggests a significant increase in transmural heterogeneity of action potentials, which may lead to abnormal ventricular firing similar to Brugada syndrome.

[0091] Figure 7 Protein-protein interaction analysis using STRING revealed that the proteins most closely related to the function of SCN2B were, in descending order: SCN1A (strongest) > SCN5A > SCN1B; and the proteins most closely related to the function of SCN4B were, in descending order: SCN5A (strongest) > ANK2 > SCN2A. Figure 7A-7B). Protein structures predicted by the AlphaFold database are shown in... Figure 7 C-7F shows the effects of these mutations on protein structure and its interactions with other amino acids. Notably, the R28Q and Y69H variations in SCN2B have a significant impact on protein structure. R28 in the WT protein does not form hydrogen bonds, but after mutation to glutamine (Q28), a hydrogen bond with proline 26 (P26) is added. Figure 7 C). Similarly, Y69 forms hydrogen bonds with E78 and G123, while its variant H69 loses the hydrogen bond with G123, which may affect protein stability to some extent. Figure 7 D). No significant conformational or amino acid interaction changes were observed for SCN2B-P210L and SCN4B-T211M. Figure 7 E, 7F).

[0092] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of reagents for detecting SCN4B gene mutations in the preparation of products for detecting hereditary arrhythmias, characterized in that, The detection product is used for early diagnosis of hereditary arrhythmias, and the reagent is used to detect the T211M mutation in the SCN4B gene.

2. The application according to claim 1, characterized in that, The full exon sequence of the SCN4B gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The reagent is also used for the combined detection of R28Q, Y69H, and P210L mutations in the SCN2B gene, the whole exon sequence of which is shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The reagents include probes for detecting SCN4B gene mutations.

5. The application according to claim 4, characterized in that, The probe can specifically recognize the T211M mutation in the SCN4B gene.

6. The application according to claim 1, characterized in that, The testing products include formulations, gene chips, or reagent kits.

7. The application according to claim 6, characterized in that, The detection product is a PCR kit, which contains primers for amplifying the SCN4B gene fragment, DNA polymerase, dNTPs, and PCR reaction buffer.

8. The application according to claim 1, characterized in that, The test samples for the testing products are peripheral blood, oral swabs, or myocardial biopsy tissue.

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