WO2018182346A1 - Composition for diagnosing or predicting drug-induced long qt syndrome, kit, and method for diagnosing or predicting drug-induced long qt syndrome using same - Google Patents

Abstract

The present invention pertains to: a composition by which drug-induced long QT syndrome (diLQTS) can be diagnosed or predicted through E409Q, which is a novel SNTA1 mutation with respect to arrhythmia, more particularly to arrhythmia occurring in drug-induced long QT syndrome patients; a kit; and a diagnosis/prediction method.

Description

Compositions, kits, and methods for diagnosing or predicting drug-induced QT prolongation syndrome

The present invention relates to compositions, kits for diagnosing or predicting drug-induced QT prolongation syndrome, and methods for diagnosing or predicting drug-induced QT prolongation syndrome using the same.

Drug-induced QT prolongation syndrome (diLQTS) is an acquired disease, mostly caused by drug block of I _Kr , such as antiarrhythmic agents. Recent studies show that sensitivity to diLQTS is increased in certain individuals because of reduced "repolarization" due to asymptomatic mutations in the two most common congenital QT prolongation syndrome loci, KCNH2 or KCNQ1, that encode K ⁺ channels. Addition of I _Kr blocking drugs to this population may increase late cardiac sodium current (I _Na-L ), thereby increasing the action potential (AP), thus increasing the QT interval associated with fatal deep vein tachycardia. Increasing I _Na-L due to drug-induced phosphoinositide 3-kinase (PI3K) signaling may also contribute to QT prolongation. However, the cardiac Na ⁺ channel Na _V 1.5 is encoded and the third most common variant of the cLQT locus SCN5A is less relevant to diLQTS.

1-syntrophin(SNTA1)은 디스트로핀 관련 단백질이며, 전압으로 개폐되는 Na⁺ 채널의 강력한 조절 인자이다. SNTA1은 모공 형성

-소단위의 C 말단과의 상호작용을 통해 Na_V1.5 채널 거대분자 복합체를 구성한다. SNTA1은 postsynaptic density protein-95 / disc large / zona occludens-1(PDZ) 도메인과 2개의 pleckstrin 상동성(PH1과 PH2) 도메인, 및 syntrophin(SU) 도메인을 포함한다. SNTA1의 PDZ 도메인은 Na_V1.5 COOH 말단의 마지막 3 개 아미노산(세린-이소류신-발린)으로 구성된 PDZ 결합 모티프와 상호 작용한다. SNTA1은 또한 원형질막 Ca-ATPase(PMCA) 4b와 상호작용하여 세 단백질의 복합체를 형성한다. 이러한 복합체는 뉴런 산화 질소 합성 효소(nNOS)를 억제하여 NOS 매개 NO 생산을 감소시킬 수 있다. 이러한 상호작용의 생리학적 및 임상적 관련성은, SNTA1의 PH2 도메인 내의 변이체(A390V)의 LQTS 환자에서 SNTA1과 PMCA4b 사이의 결합을 붕괴시켰던 이전의 동정에 의해 강조되었으며, 이로 인해 nNOS의 억제가 완화되고, 국부적으로 강조된 NO 생산에 의해 매개되는 Na_V1.5의 S-니트로실화를 통해 증가된 병원성 I_Na-L을 야기했다. 증가된 I_Na-L은 AP 지속 기간을 연장시키고 재분극 유지를 줄임으로써 심장 부정맥을 유발한다.Coded by the SNTA1 gene

1-syntrophin (SNTA1) is a dystrophin-associated protein and a potent regulator of voltage-gated Na ⁺ channels. SNTA1 forms pores

Interaction with the C terminus of the subunit forms a Na _V 1.5 channel macromolecular complex. SNTA1 comprises a postsynaptic density protein-95 / disc large / zona occludens-1 (PDZ) domain, two pleckstrin homology (PH1 and PH2) domains, and a syntrophin (SU) domain. The PDZ domain of SNTA1 interacts with a PDZ binding motif consisting of the last three amino acids (serine-isoleucine-valine) at the Na _V 1.5 COOH terminus. SNTA1 also interacts with plasma membrane Ca-ATPase (PMCA) 4b to form a complex of three proteins. These complexes can inhibit NOS mediated NO production by inhibiting neuronal nitric oxide synthase (nNOS). The physiological and clinical relevance of this interaction is highlighted by previous identifications that disrupted the binding between SNTA1 and PMCA4b in LQTS patients of the variant (A390V) within the PH2 domain of SNTA1, thereby relieving the inhibition of nNOS and , S-nitrosylation of Na _V 1.5 mediated by locally highlighted NO production resulted in increased pathogenic I _Na-L . Increased I _Na-L causes cardiac arrhythmias by prolonging AP duration and reducing repolarization maintenance.

Conventionally, no mutation of SNTA1 has been reported in diLQTS patients, but we have discovered a novel SNTA1 strain (E409Q) found in diLQTS patients, and investigated whether the SNTA1 mutation is pathogenic by increasing I _Na-L . Was completed.

It is an object of the present invention to provide compositions, kits and methods for diagnosing or predicting drug-induced QT prolongation syndrome via E409Q, a novel SNTA1 mutation associated with drug-induced QT prolongation syndrome.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the invention, consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, comprising a first primer set for specifically detecting the SNTA1 gene mutation, there is provided a composition for diagnosing or predicting arrhythmia.

According to one side, the arrhythmia may occur in a drug-induced Long QT Syndrome (diLQTS) patients.

According to one side, consisting of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, it may further comprise a second primer set to specifically detect the SNTA1 gene mutation.

According to one side, the SNTA1 gene mutation may increase late sodium current (I _Na-L ).

According to one side, the SNTA1 gene mutation may be the 1168th base of SEQ ID NO: 5 is substituted from G to T, the 1225th base is substituted from G to C.

According to another embodiment of the present invention, there is provided a kit for diagnosing or predicting arrhythmia, comprising a composition according to an embodiment of the present invention.

According to another embodiment of the present invention, the method comprises: extracting a biological sample; Amplifying the sample using a first primer set consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2; And identifying whether the SNTA1 gene is mutated; Including, an arrhythmia diagnosis or prediction method is provided.

According to one side, the amplifying the sample, amplifying using a second primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4; may further comprise a.

According to one side, the arrhythmia may occur in a drug-induced Long QT Syndrome (diLQTS) patients.

The composition for diagnosing or predicting the prolonged syndrome of drug-induced QT of the present invention can be diagnosed in advance by examining whether the ATAV or E409Q mutation of the SNTA1 gene in the subject is diagnosed, and thus whether or not a specific drug is taken, and thus the sudden death that can be caused by the drug The risk can be prevented.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the results of analysis for patients with drug-induced QT prolongation syndrome including E409Q mutation in SNTA1. 1A shows the electrocardiogram (ECG) of a subject during the post cardiac arrest period. FIG. 1B shows the electrocardiogram (ECG) of the subject for 2 months after discontinuation of heart attack. Figure 1c shows a family tree of the initiator. Arrows indicate the start of the p.E490Q mutation. 1D shows interspecies sequence conservation for normal versus A390V and E409Q in SNTA1.

2 shows electrophysiological data of Na _V 1.5 in HEK293T cells expressing WT or SNTA1 mutations simultaneously with PMCA4b, nNOS. FIG. 2A shows representative traces of inward Na ⁺ currents for the three groups under test. 2B shows the IV curve. 2c shows activation (G / Gmax) values. 2d shows the deactivation (I / Imax) values. 2E shows the recovery (P2 / P1) value.

3 shows late Na ⁺ currents in HEK293T cells. 3A shows representative late Na + currents with WT and SNTA1 mutations. Figure 3b A390V ^- shows the result of both SNTA1 and E409Q-SNTA1 increase I _Na-L significantly in HEK293T cells as compared to WT-SNTA1.

4 shows electrophysiological data of Na ⁺ currents in adult rat cardiomyocytes infected with WT or SNTA1 mutations. FIG. 4A shows representative traces of inward Na ⁺ currents for the three groups under test. 4B shows the IV curve. 4c shows the activation (G / Gmax) values. 4D shows the deactivation (I / Imax) values. 4E shows the recovery (P2 / P1) value.

FIG. 5 shows late Na ⁺ currents in adult rat cardiomyocytes infected with adenovirus expressing wild type (WT) or one of two SNTA1 mutations. 5A shows representative late Na ⁺ currents with WT and SNTA1 mutations. Figure 5b shows the result of E409Q-SNTA1 significantly increased I _Na-L compared to WT-SNTA1.

6 schematically shows the mechanism of Syntrophin mutations and late sodium currents. According to FIG. 6, the variant of SNTA1 causes a breakdown of binding to PMCA4b and exhibits an inhibitory action of nNOS, thereby increasing I _Na-L through S-nitrolation of Na _V 1.5.

7 is an image confirming the nucleotide sequence of the A390V and E409Q mutation site of SNTA1.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

The present inventors have made diligent efforts to establish a reliable genetic diagnosis method for the disease by searching for mutations specific to arrhythmias in patients with drug-induced QT prolongation syndrome. As a result, late sodium currents in E409Q, a novel mutation of the SNTA1 gene, were identified. Sodium Current, I _Na-L ) was confirmed to increase. This has led to the discovery of novel pathogenic mutations on the SNTA1 gene, which are conventionally unknown as mutations over sodium channels.

According to one embodiment of the invention, consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, comprising a first primer set for specifically detecting the SNTA1 gene mutation, there is provided a composition for diagnosing or predicting arrhythmia. The first set of primers can detect the E409Q mutation of SNTA1.

According to one side, the arrhythmia may occur in a drug-induced Long QT Syndrome (diLQTS) patients.

As used herein, the term "QT prolongation syndrome" is a congenital due to genetic abnormality, which is a serious disease that has an unexplained prolonged QT time on ECG and can cause fainting and sudden cardiac death by ventricular fibrillation. , Depending on the electrolyte and the drug can be distinguished. After a heartbeat, the heart's electrical system recharges itself for the next heartbeat, which requires longer time than a normal person with a cardiac interval syndrome, which causes an abnormally fast arrhythmia called torsade de points ( arrhythmia) comes. When these arrhythmias come, the heart can't get enough blood, and the brain doesn't get enough oxygen, causing oxygen starvation to become unconscious or die.

According to one side, consisting of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, it may further comprise a second primer set to specifically detect the SNTA1 gene mutation. The second set of primers can detect the A390V mutation of SNTA1.

According to one side, the SNTA1 gene mutation may increase late sodium current (I _Na-L ).

According to one side, the SNTA1 gene mutation may be the 1168th base of SEQ ID NO: 5 is substituted from G to T, the 1225th base is substituted from G to C. Referring to FIG. 7, the A309V mutant is replaced with amino acid No. 390 from Alanine to Valine, and the E409Q mutation is substituted with amino acid 409 from Glutamic Acid to Glutamine. .

According to another embodiment of the present invention, there is provided a kit for diagnosing or predicting arrhythmia, comprising a composition according to an embodiment of the present invention.

According to another embodiment of the present invention, the method comprises: extracting a biological sample; Amplifying the sample using a first primer set consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2; And identifying whether the SNTA1 gene is mutated; Including, an arrhythmia diagnosis or prediction method is provided. The amplifying the sample may further include amplifying the second primer set including the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

Gene amplification using the first and second primers of the present invention can be carried out through various amplification reactions known in the art, such as polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR), Ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-sustaining sequence replication, nucleic acid sequence based amplification, etc. Including but not limited to.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Subcloning and Adenovirus Production

Prepare cDNAs of SNTA1 (Genebank accession no. NM_003098.2) of pIRES2EGFP and nNOS (Genebank accession no. NM_052799.1) of pcDNA3.1, and submerge PMCA4b (Genebank accession no. AY560895) to pcDNA3.1 (Addgene) Cloned.

Human SNTA1 plasmid vectors were mutated using Quickchange II Site-Directed Mutagenesis (Agilent Technologies, Santa Clara, Calif.) And the following primers. The underlined portions in the following primers indicate variations.

Next, each of these constructs was subcloned into a pAdRFP adenovirus shuttle vector, followed by polymerase chain reaction and bacterial transformation. The AdEasy system (Agilent Technologies, Santa Clara, Calif.) Was used to generate WT- SNTA1 and mutant viruses, and adenovirus plasmids were packaged in HEK293 cells. After several freeze / thaw cycles, the recombinant virus was isolated, further amplified, and purified and concentrated using Vivapure AdenoPACK 20 (Sartorius Stedim Biotech, Goettinggen, Germany). Virus titers were determined and used at 50-200 infectivity (MOI). All structures were confirmed by sequence analysis.

HEK293T Cell Transformation and Electrophysiology

HEK293T cells at a 4: 4: 4: 1 ratio of pIRES2EGFP plasmid vector expressing tetrodotoxin (TTX) sensitive Na _V 1.5, nNOS, PMCA4b and human wild type (WT) or SNTA1 mutations (A390V and E409Q). Were transfected (Lipofectamine 2000, Life Technologies). At this time, the cells were used after incubating at 37 ° C. for 2 to 3 days, and the transfected cells were identified using green fluorescent protein (GFP).

이었다. 표준 스텝 펄스 전압(Standard step-pulse voltages)은 Axopatch 200B 증폭기로 pClamp 9.0 소프트웨어(Axon Instruments)를 사용하여 생성하였다. 전류를 5kHz에서 필터링하고 아날로그-디지털 인터페이스(Digidata 1322A, Axon Instruments)를 사용하여 디지털화 하였다. 전류 진폭 데이터 및 정상 상태 활성화의 전압 의존성을 측정하기 위해, 전류는 -120mV의 유지 전위에서 50ms 펄스에 의해 추출하여 -100mV와 + 60mV 사이의 전위를 5mV 단위로 테스트하였다. 전류 밀도(pA/pF)는 세포 용량(cell capacitance)에 대한 표준화에 의해 계산하였다. 컨덕턴스(G)는 구동력(V_m-V_r)에 의한 각 전압 단계에 대해 피크 전류를 나누고, 피크 컨덕턴스(G_max)를 정규화함으로써 계산하였다. 데이터는

로 표시되는 볼츠만 함수(Boltzmann function)를 이용하여 산출하였다. 상기 함수에서 V_1/2는 Na_v1.5 채널 활성화 시의 절반에 해당하는 전압이고,

는 기울기 계수이며, Vm은 막 전위이다. 정상상태 비활성화 곡선을 생성하기 위해 표준 2-펄스 프로토콜을 사용하였다: -120mV의 유지 전위에서 세포를 -140mV 및 -20mV 사이에서 변화하는 500-ms 프리컨디셔닝 전위까지 단계적으로 처리한 다음(prepulse), -40mV까지 20 ms 테스트 펄스를 수행하였다. After 48-72 hours of transfection, Na ⁺ currents were recorded using a whole-cell voltage-clamp technique at room temperature (20-22 ° C.). Bath solution (300mosm, mM unit) containing NaCl 120, TEA-Cl 20, KCl 5.4, CaCl ₂ 1.8, MgCl ₂ 1, HEPES 10, D (+)-glucose 10 was pH-based using NaOH. Adjusted to 7.4. Pipette solution (290-295mosm, mM unit) containing CsCl 50, CsF 30, L-aspartic acid 50, EGTA 5, HEPES 10, NaCl 10 was adjusted to pH 7.3 using CaOH. Osmolarity of all solutions was adjusted using sucrose. The resistance of the electrode is 2-4M

It was. Standard step-pulse voltages were generated using pClamp 9.0 software (Axon Instruments) with an Axopatch 200B amplifier. The current was filtered at 5kHz and digitized using an analog-digital interface (Digidata 1322A, Axon Instruments). To measure the current amplitude data and the voltage dependence of steady state activation, the current was extracted by 50 ms pulses at a holding potential of -120 mV and tested for potentials between -100 mV and +60 mV in 5 mV increments. Current density (pA / pF) was calculated by normalization to cell capacitance. Conductance (G) was calculated by dividing the peak current for each voltage step by driving force (V _m -V _r ) and normalizing the peak conductance (G _max ). The data is

It was calculated using the Boltzmann function represented by. In the above function, V _1/2 is the voltage corresponding to half of the Na _v 1.5 channel activation,

Is the slope coefficient and V m is the membrane potential. A standard two-pulse protocol was used to generate a steady state deactivation curve: cells were processed stepwise at a holding potential of -120 mV up to a 500-ms preconditioning potential that varied between -140 mV and -20 mV, and then A 20 ms test pulse was performed up to -40 mV.

형태의 볼츠만 함수로 산출하였다. 상기 함수에서 V_1/2는 Na_v1.5 채널 활성화 시의 절반에 해당하는 전압이고,

는 기울기 계수이며, Vm은 막 전위이다. 불활성화로부터의 회복은 2개의 지수 함수를 이용하여 산출하였다:

상기 함수에서 A 및

는 각각 진폭과 시간 상수를 의미한다. I_NA-L은 탈분극 개시 후 145-150ms 사이의 평균으로서 -120mV에서 -10mV까지 200ms 탈분극으로 결정하고,

TTX(Abcam Biochemicals)의 존재 및 부존재 하에서 기록된 전류의 디지털 감산 이후의 피크 전류로 기록하였다. 곡선 근사(curve fitting) 및 데이터 분석은 Clampfit 10.5 소프트웨어(Axon Instruments) 및 Origin 9.1(OriginLab Corporation)를 사용하여 수행하였다.Current I is normalized to Imax,

The Boltzmann function of the form was calculated. In the above function, V _1/2 is the voltage corresponding to half of the Na _v 1.5 channel activation,

Is the slope coefficient and Vm is the membrane potential. Recovery from inactivation was calculated using two exponential functions:

In the function, A and

Denote amplitude and time constant, respectively. I _NA-L is the average between 145-150ms after the initiation of depolarization and is determined as 200ms depolarization from -120mV to -10mV

The peak current after digital subtraction of the recorded current in the presence and absence of Abtx Biochemicals (TTX) was recorded. Curve fitting and data analysis were performed using Clampfit 10.5 software (Axon Instruments) and Origin 9.1 (OriginLab Corporation).

Isolation, Culture and Adenovirus Infection of Mature Rat Ventricular Muscle Cells

m 나일론 메쉬를 통해 여과하고 2 분 동안 300rpm에서 원심분리 하였다. 상기 세포를 5 mg/ml의 소혈청 알부민(BSA)과 함께 관류 용액에 재현탁시켜 효소를 냉각시켰다. 칼슘 내성은 CaCl₂를 1mM의 최종 농도로 서서히 첨가하여 수행하였다. Earle’s salt, L-glutamine, 10mM BDM, 5% 소태아혈청(Life Technologies) 및 1% 페니실린/스트렙토마이신을 포함하는 최소배양액(MEM)의 평판 배지 내 laminin 코팅된 커버슬립 상에 세포를 도말하고 세척하였다. 바이러스를 배지에 재현탁하고 평판 배지를 아데노바이러스가 첨가된 배양 배지로 교체하였다. 배양 배지는 Earle’s Salts, L-글루타민, BSA 0.1 mg/ml, BDM 10 mM, 1X insulin-selenium- transferrin supplement(Gibco), creatine 5 mM, taurine 5 mM, L-carnitine 2 mM, 및 blebbistatin 25

M(Toronto Research Chemicals)을 포함하는 MEM 배지를 사용하였다. 95% O₂ / 5% CO₂조건 에서 30분 이상 모든 용액에 산소를 공급하였다. 감염 36-48시간 경과 후 RFP 형광을 통해 세포를 확인하였다. 전기생리학을 통해 막대 모양의 줄무늬 세포를 관찰하였다. Animals were administered according to the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals . This study was approved by the Duke University Animal Care and Welfare Committee. Cardiomyocytes were isolated and cultured from 6-8 week old rats (Charles River Laboratories, Wilmington, Mass.). Rats were stored in cages equipped with sterilization chambers with controlled temperature and humidity ranges (20-26 ° C., 30-70% humidity, 10-15 fresh air supply per hour). Time-controlled lighting systems were used to ensure regular weekly cycles, and the timer performance was periodically checked to provide adequate cycling. Animals were anesthetized with anticoagulants containing tribromoethanol (250 mg / kg, intraperitoneal injection) and heparin according to the Duke University Animal Care & Use Program guidelines for general anesthesia in rats. The heart was then removed and inserted into the aorta using the Langendorff device (Radnoti Glass Technology) to perfusion the heart back into the line for about 10 minutes. NaCl 112, KCl 5.4, NaH2PO4H2O 1.7, NaHCO3 4.2, MgCl6H2O 1.63, HEPES 20.04, D (+)-glucose 5.4, taurine 10, L-carnitine 2, creatinine 2.3, glucose 5.4, taurine 10, L-carnitine The heart was first perfused with a basal solution containing 2, creatine 2.3, 2,3-butanedione monoxime (BDM) 10 (mM, unless otherwise specified) from sigma. After 5 minutes, 150 U / ml type II collagenase (Worthington) was converted to the base solution, the heart was perfused and the heart was removed from Langendorff. Both ventricles were chopped into small pieces and then crushed with enzyme solution until all cell masses were broken. Aseptic solution 190

Filtered through m nylon mesh and centrifuged at 300 rpm for 2 minutes. The cells were resuspended in perfusion solution with 5 mg / ml bovine serum albumin (BSA) to cool the enzyme. Calcium resistance was carried out by the slow addition of CaCl ₂ to a final concentration of 1 mM. Plate and wash cells on laminin-coated coverslips in plate media of minimal culture medium (MEM) containing Ear's salt, L-glutamine, 10 mM BDM, 5% fetal bovine serum (Life Technologies) and 1% penicillin / streptomycin It was. The virus was resuspended in medium and the plate medium was replaced with culture medium with adenovirus added. Culture medium is Earle's Salts, L-Glutamine, BSA 0.1 mg / ml, BDM 10 mM, 1X insulin-selenium-transferrin supplement (Gibco), creatine 5 mM, taurine 5 mM, L-carnitine 2 mM, and blebbistatin 25

MEM medium containing M (Toronto Research Chemicals) was used. Oxygen was supplied to all solutions for at least 30 minutes at 95% O ₂ /5% CO ₂ . Cells were identified by RFP fluorescence 36-48 hours after infection. Sticky striped cells were observed through electrophysiology.

Cardiomyocyte Electrophysiology

로 표시되는 볼츠만 함수(Boltzmann function)를 이용하여 산출하였다. 상기 함수에서 V_1/2는 Nav1.5 채널 활성화 시의 절반에 해당하는 전압이고,

는 기울기 계수이며, V_m은 막 전위이다. 정상상태 비활성화 곡선을 생성하기 위해 표준 2-펄스 프로토콜을 사용하였다: -120mV의 유지 전위에서 세포를 -140mV 및 -20mV 사이에서 변화하는 500-ms 프리컨디셔닝 전위까지 단계적으로 처리한 다음(prepulse), -40mV까지 20 ms 테스트 펄스를 수행하였다. 전류(I)는 Imax로 표준화하고,

형태의 볼츠만 함수로 산출하였다. 상기 함수에서 V1/2는 Nav1.5 채널 활성화 시의 절반에 해당하는 전압이고,

는 기울기 계수이며, Vm은 막 전위이다. 불활성화로부터의 회복은 2개의 지수 함수를 이용하여 산출하였다:

상기 함수에서 A 및

는 각각 진폭과 시간 상수를 의미한다. I _NA-L은 탈분극 개시 후 190-200ms 사이의 평균으로서 -120mV에서 -40mV까지 200ms 탈분극으로 결정하고, 30

TTX(Abcam Biochemicals)의 존재 및 부존재 하에서 기록된 전류의 디지털 감산 이후의 피크 전류로 기록하였다. 특정 그룹에 대해서는 조사자로부터 시야를 가리고 결과를 측정하였다. 곡선 근사(curve fitting) 및 데이터 분석은 Clampfit 10.5 소프트웨어(Axon Instruments) 및 Origin 9.1(OriginLab Corporation)을 사용하여 수행하였다.As described above, Na ⁺ currents were recorded using whole-cell voltage-clamps in cardiomyocytes. After 36-48 hours from infection of adenovirus on adult cardiomyocytes, voltage clamp experiments were performed at room temperature (22-24 ° C.). Bath (Tyrode) solution containing NaCl 140, KCl 5.4, CaCl ₂ 1, MgCl ₂ 6H ₂ O 1, HEPES 5, glucose 10 (mM unit) was adjusted to pH 7.2 using NaOH. Recording solution (mM) containing NaCl 20, HEPES 20, CsCl 55, CaCl2 1, MgCl2 1, CsOH 10, 4-aminopyridine 2, D (+)-glucose 10, CdCl2 0.5, TEA-Cl 50 after cell rupture Unit) and adjusted to pH 7.35 with HCl. The internal solution (mM unit) included NaCl 5, CsF 120, HEPES 5, EGTA 10, GTP-Na sulfate 0.5, TEA-Cl 20 and adjusted to pH 7.35 using CsOH. Osmolarity of all solutions was adjusted to ˜300 mOsm using sucrose. Records were filtered at 5 kHz and digitally sampled at 20 kHz. Pulse protocol period was maintained at 3 seconds for complete Na ⁺ channel recovery. Current amplitude data of each cell was normalized to cell capacity (current density, pA / pF). To determine the steady state activation voltage dependence, the current was extracted by a 50 ms pulse at a holding potential of -120 mV and tested for potentials between -100 mV and +60 mV in 5 mV increments. Sodium conductance (G) was calculated by dividing the peak current for each voltage step by driving force (Vm-Vr) and normalizing the peak conductance (Gmax). The data is

It was calculated using the Boltzmann function represented by. In the above function, V _1/2 is the voltage corresponding to half of the Nav1.5 channel activation,

Is the slope coefficient and V _m is the membrane potential. A standard two-pulse protocol was used to generate a steady state deactivation curve: cells were processed stepwise at a holding potential of -120 mV up to a 500-ms preconditioning potential that varied between -140 mV and -20 mV, and then A 20 ms test pulse was performed up to -40 mV. Current I is normalized to I max,

The Boltzmann function of the form was calculated. In the function, V1 / 2 is half the voltage at the Nav1.5 channel activation,

Is the slope coefficient and Vm is the membrane potential. Recovery from inactivation was calculated using two exponential functions:

In the function, A and

Denote amplitude and time constant, respectively. I _NA-L is the average between 190 and 200 ms after the initiation of depolarization, determined as 200 ms depolarization from -120 mV to -40 mV, 30

The peak current after digital subtraction of the recorded current in the presence and absence of Abtx Biochemicals (TTX) was recorded. For certain groups the field of view was masked from the investigator and the results measured. Curve fitting and data analysis were performed using Clampfit 10.5 software (Axon Instruments) and Origin 9.1 (OriginLab Corporation).

Statistical analysis

test는 이용하여 비교하였다.　 P≤0.05의　값을　통계적으로 유의한 것으로 간주하였다.　 통계 분석은 SPSS Statistics 소프트웨어 버전 17.0(IBM, Armonk, NY)을 사용하여 수행하였다.Results are expressed as mean ± standard error (SEM). The statistical significance of the differences between the groups was assessed by Student's t- test. For parametric tests for normality, nonparametric Mann-Whitney

The test was compared using. Values of P ≦ 0.05 were considered statistically significant. Statistical analysis was performed using SPSS Statistics software version 17.0 (IBM, Armonk, NY).

result

Case report

Sudden cardiac death or a healthy 36-year-old man without LQTS family history suffered a heart attack during exercise. The men's taking medications at the time of a heart attack included amitrifthilin, pseudoephedrine and pamotidine. His first rhythm was ventricular fibrillation and was successfully revived by external CPR. Coronary angiography showed no evidence of coronary artery disease. Cardiac MRI showed 65% EF without evidence of right ventricular dysplasia. It was noteworthy that electrocardiograms (ECGs) after heart failure showed extended QT intervals (repeated QTc measurements> 480 ms; longest was 597 ms) (FIG. 1A). Electrocardiogram results obtained during all follow-up periods after heart failure showed normal QT intervals after stopping amitriphthyline, pseudoephedrine and pamotidine (all QTc measurements <440 ms) (FIG. 1B).

-차단제 치료를 처방받았고, 이식 가능한 심장제세동기(ICD)를 이식받았다. 그는 격렬한 운동을 재개한 후 다형성심실성빈맥에 대한 ICD 쇼크를 1회 받았으나, 간헐적으로

-차단제 치료를 받았다.Said patient

He was prescribed blocker therapy and had an implantable cardiac defibrillator (ICD). He received a single ICD shock for polymorphic ventricular tachycardia after resuming vigorous exercise, but intermittently

-I received a blockade treatment.

SNTA1  Genetic testing and sequence homology for mutations

Genetic tests using 30-gene panel arrhythmias, it has been found a new heterozygous (p.Glu409Gln, E409Q c.1225 G> C) in SNTA1. The family tree is shown in Figure 1c. These mutations are within highly conserved residues throughout FIG. 1D and are similar to the A390V SNTA1 mutations previously reported and used as positive controls with respect to LQTS. E409Q SNTA1 mutations were not present in the database including Exome Aggregation Consortium (ExAC), NHLBI ESP Exome Variant Server (EVS) and 1000 Genomes Project. Various in-silico analyzes predicted that the SNTA1 mutation (E409Q) would be pathogenic: PolyPhen-2 (prediction = damage, score = 1.000), MutationTaster2 (prediction = disease incidence, probability = 0.999) alc SIFT (prediction = Damage, score = 0).

We recorded voltage-dependent Na ⁺ currents in HEK293T cells that transiently and simultaneously express components of the Na ⁺ channel macromolecular complexes essential for regulation by SNTA1 . In particular, we used wild-type SNTA1 or one of two SNTA1 mutations to express human Na _V 1.5 (C373Y mutant represents a TTX sensitive channel), nNOS and PMCA4b. Previous studies have reported that TTX sensitive mutations do not affect any permeation properties. Table 1 shows the electrophysiological summary data for three groups in HEK293T cells. The number of cells analyzed for each parameter in Table 1 below is shown in parentheses, and I _Na-L refers to late Na currents. In addition, * indicates that the p-value is less than 0.05 compared to WT-SNTA1.

는 E409Q-SNTA1에서만 유의적으로 감소 하였다(p <0.001). 야생형과 돌연변이 간 2-펄스 프로토콜을 이용한 불활성화 속도에서 유의적인 차이가 나타나진 않았으나, A390V-SNTA1에서 WT-SNTA1(p=0.035)과 비교하여 회복 속도가 유의적으로 지연되었다(도 2d). 돌연변이에만 초점을 맞추면 활성화, 불활성화 및 회복에 대한 모든 파라미터에서 유의적인 차이가 나타나지 않았다.Total cell currents (FIG. 2A) and IV curves (FIG. 2B) show that mutations compared to wild type SNTA1 did not affect peak I _Na current density and activation rate (FIG. 2C). Inactivation

Was significantly decreased only in E409Q-SNTA1 (p <0.001). There was no significant difference in inactivation rate using the two-pulse protocol between wild type and mutant, but the recovery rate was significantly delayed compared to WT-SNTA1 (p = 0.035) in A390V-SNTA1 (FIG. 2D). Focusing on mutations alone showed no significant differences in all parameters for activation, inactivation and recovery.

M)의 투여로 백그라운드를 제거하였다. I_NA-L(피크 전류의 %)는 야생형 SNTA1에 비해 2개의 돌연변이 모두에서 유의적으로 증가 하였다(야생형에서 0.58±0.10 vs A390V-SNTA1에서 0.90±0.11, p=0.048; vs. E409Q-SNTA1에서 0.88±0.07, p = 0.023)(도 3b). A390V-SNTA1과 E409Q-SNTA1 간 I_Na-L는 유의적인 차이가 없었다(p=0.903).I _Na-L was measured using a long depolarization pulse (200 ms at -10 mV from a holding potential of -120 mV) and TTX (1

The background was removed by the administration of M). I _NA-L (% of peak current) was significantly increased in both mutations compared to wild-type SNTA1 (0.58 ± 0.10 vs wild type, 0.90 ± 0.11 in A390V-SNTA1, p = 0.048; vs. E409Q-SNTA1). 0.88 ± 0.07, p = 0.023) (FIG. 3B). There was no significant difference in I _Na-L between A390V-SNTA1 and E409Q-SNTA1 (p = 0.903).

Biophysical Characteristics of Sodium Current in Adult Rat Cardiomyocytes

We recorded voltage gated Na ⁺ currents in adult rat cardiomyocytes cultured for 36-48 hours after infection with wild type or one of two SNTA1 mutations. Because the patient is a heterozygous for SNTA1 mutation, the rat cardiomyocyte expresses wild type or mutation without digesting endogenous Snta1 and thus more accurately repeats the condition of the patient where both wild type and mutation are present. Table 2 shows the electrophysiological summary data for three groups in adult rat cardiomyocytes. The total number of mice used in the study was 11. Experiments were performed on cells derived from various isolates. Some cells were recorded from single rat isolates. The number of cells analyzed for each parameter is shown in parentheses and I _Na-L refers to late Na currents. In addition, * indicates that the p-value is less than 0.05 compared to WT-SNTA1.

Total cell current is shown in FIG. 4A. The IV curve (FIG. 4B) shows that the mutation did not affect the I _NA current density (WT-SNTA1 vs. A390V-SNTA1, p = 0.895 vs. E409Q-SNTA1, p = 0.929). In addition, there was no significant difference in voltage between variants (A390V-SNTA1 vs. E409Q-SNTA1, p = 0.94). The dynamics of activation and steady state inactivation of wild type and mutations are shown in FIGS. 4C and 4D. There was no significant difference in the dynamics of activation and steady-state inactivation between wild-type and SNTA1 mutations. Compared to WT-SNTA1, the recovery rate from inactivation appeared to be slightly delayed in E409Q-SNTA, but there was no statistical significance (FIG. 4E).

M)를 배스(bath)에 적용한 후 백그라운드를 제거하였다. 대표적 트레이스(trace)를 도 5a에 나타내었고, 데이터를 표 2에 요약하였다. WT-STNA1에 비해 I_NA-L(피크 전류의 %)은 E409Q 돌연변이에서 유의적으로 증가되었지만 A390V 돌연변이에서는 유의적이지 않았다(WT-SNTA1에서 0.49±0.14 vs. A390V-SNTA1에서 0.94±0.23, p=0.099; vs E409Q-SNTA1에서 1.12±0.24, p=0.019)(도 5b). A390V-SNTA1과 E409Q-SNTA1 사이에 I_NA-L에는 유의적인 차이가 없었다(p = 0.903).I _Na-L was measured using a long depolarization pulse (200 ms at -40 mV from a holding potential of -120 mV) and TTX (30

M) was applied to the bath and the background was removed. Representative traces are shown in FIG. 5A and data is summarized in Table 2. Compared with WT-STNA1, I _NA-L (% of peak current) was significantly increased in E409Q mutations but not significantly in A390V mutations (0.49 ± 0.14 vs. WT-SNTA1 vs. 0.94 ± 0.23, p in A390V-SNTA1) = 0.099; vs. 1.12 ± 0.24, p = 0.019 in E409Q-SNTA1) (FIG. 5B). There was no significant difference in I _NA-L between A390V-SNTA1 and E409Q-SNTA1 (p = 0.903).

Key result

We have found that SNTA1 affects I _Na-L in cardiomyocytes of adult rats. We have demonstrated that the gain of function of the novel SNTA1 variant E409Q causes diLQTS by increasing I _Na-L . Overall, the effect of the E409Q variation on the Na ⁺ current is generally similar to that of the A390V variation, the original LQT variation described in SNTA1.

Late sodium currents with syntrophin mutations

Na _V 1.5 channel interaction protein (NaChIP) complexed with nNOS and PMCA4b has been established. Mutations induce the function acquisition regulation of NaV1.5 (increased I _Na-L ) and cLQTS. Ueda et al. Reported that mutations in the PH2 domain of SNTA1 (A390V) disrupted binding to PMCA4b, inhibiting nNOS, leading to S-nitrosylation of NaV1.5 and an increase in I _Na-L . Novel mutants are disclosed herein and E409Q is outside the PH2 region. Thus, E409Q affects Na _V 1.5 currents similarly to A390V in both cardiomyocytes and E409Q affects PMCA4b interactions similar to A390V in heterogeneous systems where all major components of the macromolecular complex are present. You can check it. Thus, we concluded that the binding site for PMCA4b should extend further towards the C-terminus of PMCA4b than the PH2 domain or that the E409Q variant allosterically affects the PH2 binding domain. 6 schematically shows the resulting mechanism for the aforementioned interactions and increased I _Na-L .

Increasing I _Na-L due to SNTA1 mutations is part of the pathogenesis of cLQTS and channel pathogenic infantile death syndrome (SIDS), and mutations are indicative of the acquisition of I _Na . E409Q and A309V SNTA1 variants are wild type SNTA1. Compared with, increase I _Na-L without showing significant difference in peak I _Na .

E409Q mutations in HEK293T cells showed a reduced k inactivation, and adenovirus expression was used to confirm the electrophysiological characteristics of SNTA1 mutations in adult rat cardiomyocytes, indicating that recovery from inactivation was observed in HEK293T cells and cardiomyocytes. It was found to be different when comparing.

SNTA1: Novel Sensitive Genes for diLQTS

-서브유닛 또는 caveolin-3 및 SNTA1과 같은 NaChIP에 영향을 미치므로 I_Na-L이 증가한다. 본 실험의 환자는 diLQTS, 선천적 또는 의원성 질환으로 진단되었는데, 이는 포타슘 채널 차단으로 인한 재분극 이상으로 인한 것임이 가장 일반적으로 설명되었다. 도 1a에 도시된 노치된 T파는 I_Kr 블록 후에 관찰된 패턴과 일치한다. I_Kr(hERG 채널) 차단제, 위장운동촉진제(prokinetics) 또는 항부정맥제(antiarrhythmic)는 환자의 "재분극 유지"를 감소시키고 QT 간격을 연장할 수 있다. 본 실험의 환자는 급사 전에 3 가지 약제(심환계 항우울제(tricyclic antidepressant), 아미트리프틸린; 슈도에페드린; 및 H₂-수용체 길항제 파모티딘)에 노출되었고, 이들 중 2개(아미트리프틸린과 슈도에페드린)는 QT 간격 연장을 유발하는 것으로 알려져 있다. 즉, diLQTS이 아미트리프틸린과 슈도에페드린에 의한 재분극 유지의 감소로 악화된 cLQTS 유전자좌(SNTA1)에서의 돌연변이로 인한 유전적 감수성에 기인하는 것이라 할 수 있다. 본 실험결과는 SNTA1의 변이로 인한 민감성을 포함하도록 상기 개념을 확장한다. 병리학적인 SNTA1 변이에 관한 이전의 보고서에서, 영향받은 환자들은 약물 처치 시 갑작스런 사망 사건을 중단하고 약물 중단 후 심전도상 정상 QT 간격을 보인 인덱스 환자와 달리 cLQTS 표현형을 보였다. 또한 같은 변이를 가진 발단자(proband)의 가족 구성원도 정상 QTc 간격(그림 1C)을 나타내고 어떠한 징후도 나타내지 않았다. Voltage-gated cardiac sodium channels are known to be responsible for a subgroup of LQTSs (LQT3). The other subgroups of LQTS (LQT9, LQT10 and LQT12) are also found in Na _V 1.5.

I _Na-L increases because it affects subunits or NaChIPs such as caveolin-3 and SNTA1. Patients in this study were diagnosed with diLQTS, congenital or clinic disease, most commonly explained by repolarization abnormalities due to potassium channel blockade. The notched T wave shown in FIG. 1A is consistent with the pattern observed after the I _Kr block. I _Kr (hERG channel) blockers, prokinetics or antiarrhythmic may reduce the "repolarization" of the patient and prolong the QT interval. Patients in this study were exposed to three medications (tricyclic antidepressant, amitriptyline; pseudoephedrine; and H ₂ -receptor antagonist famotidine) prior to sudden death, and two of them (amitrifitlin and Pseudoephedrine) is known to cause prolongation of the QT interval. In other words, diLQTS may be due to genetic susceptibility due to mutations in the cLQTS locus (SNTA1), which are exacerbated by a decrease in the repolarization maintenance by amitriptyline and pseudoephedrine. The results of this experiment extend the concept to include the sensitivity due to mutations in SNTA1. In previous reports of pathological SNTA1 mutations, affected patients showed a cLQTS phenotype, unlike index patients, who stopped abrupt death on medication and showed normal QT intervals on ECG after drug discontinuation. In addition, family members of the same variant of the proband also showed normal QTc intervals (Figure 1C) and no signs.

Clinical Significance

The identification of variants in the cLQTS locus in patients suffering from diLQTS can provide advice on cascade screening and outcomes to affected family members to avoid known QT prolongation drugs. In addition, for patients suffering from diLQTS in SNTA1 or other modifications of NaChIP, especially when it is considered necessary to co-administer the QT extender, a drug targeting I _Na-L such as ranolazine is recommended. Treatment may be a reasonable strategy.

conclusion

1-syntrophin의 변이가 유전적 민감성의 잠재적 기전임을 보여줌으로써, cLQTS 내의 변종이 diLQTS를 유발할 수 있다는 개념을 K⁺ 채널 유전자좌를 넘어서 확대시킨다.In conclusion, we found that by increasing I _Na-L , a new SNTA1 variant, E409Q-SNTA1, leads to diLQTS. The data in this study are Na _V 1.5 interactivity in diLQTS patients.

By demonstrating that 1-syntrophin mutations are a potential mechanism of genetic susceptibility, we extend the notion that variants in cLQTS can induce diLQTS beyond the K ⁺ channel locus.

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

Claims (9)

Comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, comprising a first primer set to specifically detect the SNTA1 gene mutation, arrhythmia diagnostic or prediction composition. The method of claim 1, The arrhythmias occur in drug-induced Long QT Syndrome (diLQTS) patients, arrhythmia diagnosis or prediction composition. The method of claim 1, Comprising the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and further comprising a second primer set for detecting specifically the SNTA1 gene mutation, arrhythmia diagnostic or prediction composition. The method of claim 1, The SNTA1 gene mutation increases late sodium current (I _Na-L ), arrhythmia diagnostic or prediction composition. The method of claim 1, The SNTA1 gene mutation is that 1168th base of SEQ ID NO: 5 is substituted from G to T, 1225th base is substituted from G to C, arrhythmia diagnostic or prediction kit. A kit for diagnosing or predicting arrhythmia comprising the composition of any one of claims 1 to 5. Extracting the biological sample; Amplifying the sample using a first primer set consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2; And Identifying whether the SNTA1 gene is mutated; Comprising, arrhythmia diagnosis or prediction method. The method of claim 7, wherein The amplifying the sample may further include amplifying using a second primer set consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, arrhythmia diagnosis or prediction method. The method of claim 7, wherein The arrhythmia occurs in a drug-induced Long QT Syndrome (diLQTS) patient, arrhythmia diagnosis or prediction method.

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