The Genetic Molecular Mechanism Study Of Early Repolarization Associated Sudden Cardiac Death Resulted From A Calcium Channel Gene CACNA1C Mutation

BackgroundSudden cardiac death(SCD)is the natural death caused by cardiovascular diseases,the top killer of human health.Among the most common cardiovascular diseases with high risk of SCD,the inherited arrhythmia syndrome,especially in the young got an increasingly attention.Inherited arrhythmia syndromes are mainly caused by various ion channel proteins and the regulatory protein-coding gene mutations,and the heart structure and function of patients with the diseases are always normal when characterized wave morphology shown in the electrocardiogram.Under some specific conditions,malignant arrhythmias might be induced by the severe disturbances of the electrophysiological activation,and eventually cause SCD.Early repolarization(ER)is characterized by an elevation of the J point and/or ST segment in the electrocardiogram.ER is common in the general population and has been accepted as a benign electrocardiogram variant for the half century.As the development of epidemiological investigation,some people with ER had increased risk of SCD and family clustering.It is a challenge for clinicians to distinguish the ER patterns with risk of SCD or not.According to the latest expert consensus statement on the diagnosis and management of patients with inherited arrhythmia syndromes,early repolarization syndrome(ERS)is defined in the presence of patient with J-point elevation ≥1 mm in ≥2 contiguous inferior and/or lateral leads of ECG,particularly for those who accompany with arrhythmias,syncope or sudden death.In recent years,with the blooming development of molecular genetics theory and technology,the knowledge and exploration of different types of inherited primary arrhythmia syndromes has been greatly promoted.To date,cardiac ionic channel encoding gene mutations have been reported to be associated with the development of ERS,such as KCNJ8 and ABCC9(ATP sensitive potassium channels-Kir6.1 encoding genes),CACNA1 C,CACNB2b and CACNA2D1(L type calcium channel encoding genes),and SCN5A(sodium channel encoding gene).The increased outward potassium current and decreased inward calcium or sodium current has been considered to be the pathogenic basis of ERS.Unlike other inherited arrhythmia syndromes such as long QT syndrome and Brugada syndrome with significant genetic features,the pathogenic mutations of patients with ERS were mainly found in sporadic cases but rare in the family,especially in the Chinese population.There are so huge spaces existed for the genetic study of ERS in Chinese.The cascading genetic screening for the patients with obvious symptoms or a family history of SCD would high-efficiently find the pathogenic gene mutations,and further explore the pathogenesis of ERS and make more effective therapeutic strategies for individuals.Section 1 Clinical Analysis and Genetic Testing in a Large Family with Sudden Cardiac DeathIn this study,we make a comprehensive clinical evaluation for the SCD proband and his survive family members,the autopsy report of the proband was analysed in details,and screening of SCD candidate genes were done for the family to find the development rule of SCD,and potential causes of death and pathogenic gene mutations based on the clues of abnormal electrocardiogram findings,and finally we provide medical advices for the family members with high risk of SCD.Methods: 1.Clinical investigation: Our study strictly complianced with the medical ethical principles,and was approved by the ethics committee of our hospital when all the participants gave a written informed consent to be included in the study.The clinical data of the SCD victim from Jiangxi province and his relatives with high risk of SCD was collected when they consulted to our cardiology department.SCD was diagnosed according to the 2013 HRS/EHRA/APHRS expert consensus statement on the diagnosis and management of patients with inherited primary arrhythmia syndromes.The medical history,clinical examination results of electrocardiogram,echocardiography and so on in the family members were obtained.2.Autopsy investigation: The autopsy report and related pathological tissue of the deceased provided from judicial institution were abtained,and the legal representative of the deceased gave a written informed consent.3.HE staining of the proband’s myocardial tissue: The myocardial tissue in different location from the dead proband was dealed with HE staining to view the structure and morphology.4.Genetic testing: Blood samples(5 m L)were obtained from the paticipants.The whole genomic DNA was extracted from peripheral blood leukocytes by using a Relax Gene Blood DNA System.The common pathogenic genes of inherited arrhythmic syndromes were chose as candidate genes.All exons and exon-intron boundaries of the sodium ion channels encoding genes SCN5A、SCN1B、SCN3B and GPA1L,and potassium ion channels encoding genes KCNQ1、KCNH2、KCNJ8、KCNE1 、 KCNE2 and KCND3,and calcium ion channels encoding genes CACNA1C、CACNB2b and CACNA2D1,along with ANK2 and PRKAG2 were directly sequenced.400 healthy ethnically and age matched individuals was used randomly selected as controls to distinguish the mutation and single-nucleotide polymorphisms(SNPs).5.Statistical analysis: All statistics were performed by using SPSS 19.0 software.Numerical data are presented as means ± standard errors of the mean(SEM).Statistical comparisons were performed with t-test or one-way ANOVA test.P values of <0.05 was considered to be statistically difference.P values of <0.01 was considered to be statistically significant difference.Results: 1.A large family with unexplained sudden death at night was collected in our study.Five male family members,which including the proband suffered from sudden death successively during their sleep at their youth age,and the youngest death was 23 years old,oldest death was 49 years old,the mean age was 34 ± 8.4 years old.2.The autopsy report of the proband indicated that there was no pathogenic change caused by mechanical damage and asphyxia,and lack of toxic evidence.Besides,the histopathological examination of the major organ was negative,the sudden death might be cardiac original.We got some heart tissue from the proband,and the pathological section showed the morphological structure and arrangement of the cardiomyocytes was normal.So the cardiomyopathy and some other structural heart diseases,Brugada syndrome with pathological right ventricular change were not considered to be the cause of death.The proband manifested with occasional dizziness and palpitations,but without chest distress,chest pain and syncope in the past three years.His previous echocardiography was normal,but the 24 hours 3-lead Holter recorded terminal QRS notches with J point ≥1.5 mm in lead II and lead a VF.3.There was no any positive finding from other family members of the proband,including the past medical and personal history,physical examination,rest electrocardiogram,echocardiography and exercise test.However,the 24 hours-Holter of 3 family members showed the presence of early repolarization.There were most remarkable early repolarization waves of the younger brother of proband,a wide range of the limb(II,III and a VF)leads and chest(V3 ~ V5)leads revealed the J-point elevation(0.05 m V ~ 0.3 m V)and J waves,and peaked T waves in V2 ~ V4 leads.ST-segment elevation in the inferior and lateral leads was also observed in the Holter test of the proband’s son,and the daughter of his brother.4.A heterozygous missense mutation of theα1 subunit of calcium channel encoding gene CACNA1 C was identified in the family by sequencing of the candidate genes.The mutation was caused by the transition of 5747 nucleotide base A to G in exon 48,leading to the amino acids arginine replaced by glutamine at the position 1916(p.Q1916R).The mutation existed in the proband and all of his firstand secondary-degree relatives except his mother,while other family members and the sibling of his mother lacked of the mutation.In addition,five CACNA1 C gene SNPs were found,including c.2436C> T(p.D812D),c.3786C> T(p.F1262F),c.5361G> A(p.T1787T),c.5609C> T(p.T1870M)and c.5649G> A(p.P1883P),as well as an SCN5 A gene SNPs,c.3578G> A(p.R1193Q).5.The electrocardiogram parameters were compared between the CACNA1C-Q1916 R mutation carriers with or without performance of early repolarization.The heart rate corrected QT interval was shorter in the family members with early repolarization,but all in the normal range.While the QT dispersion(QTd)and Tp-Te dispersion(Tp-ed)was larger,without statistically significant difference.Conclusions: 1.In this study,we demonstrated a rare early repolarization syndrome family with strong positive family history of SCD in young male members.2.A novel mutation of CACNA1C-Q1916 R might be associated with the SCD caused by early repolarization syndrome in the family.3.The gain-of-function SNPs of SCN5A-R1193 Q might play a protective role in the SCD caused by early repolarization syndrome in the family.4.The increased transmural dispersion of repolarization parameters(QTd and Tp-ed)suggested the risk of malignant arrhythmia,were shown in the CACNA1 C mutation carriers with early repolarization.But there was no statistically significant difference when compared with the CACNA1 C mutation carriers without early repolarization.The limited sample size might be the reason.5.Other CACNA1 C gene SNPs(D812D,F1262 F,T1787T,T1870 M and P1883P)might be not the cause of SCD in this family.Section 2 Molecular Genetic and Cellular Electrophysiological Mechanisms and Drug Intervention Study of the CACNA1 C Gene Mutation in Sudden Cardiac Death Related to Early RepolarizationTo investigate the functional change and molecular mechanisms of CACNA1CQ1916 R mutation in ER related to SCD from level of tissues and cellular,and screening of effective drug treatment.Methods: 1.Immunohistochemical testing of human myocardial tissue specimen: The left ventricular of proband was used to detect the expression level and location of Cav1.2,and the healthy left ventricular was taken as a control;2.Site-directed mutation of CACNA1C-Q1916R: The classical technology of site-directed mutation was used to make the 5747 th A-to-G mutation of CACNA1 C,and excluded other mutations by sequencing;3.Transient transfection of CACNA1C: The wide type and mutant plasmid of CACNA1 C,together with the CACNB2 b and CACNA1 C were co-transfected to the human embryonic kidney 293(HEK293)cells according to the molarity 1:1:1 by transfection kit of Lipofectamine? 2000.The transfected HEK293 cells for cellular electrophysiology experiment were transfected with p EGFP-N3 of the molarity 0.3 additionally;4.Quantitative Real time PCR(q RT-PCR): The fragment sequence of wide type and mutant CACNA1 C expressed in HEK293 cells were amplified to determine the mutant effect to the expression level of total m RNA;5.Western Blot: The protein expression levels included total lysates,membrane and cytoplasmic of the wide type and mutated Ca V1.2 expressed in HEK293 cells were detected by Western Blot;6.Immunocytochemistry: The expression,location and trafficking of Cav1.2 marked by Cav1.2 specific fluorescence antibody in the wide type and mutant CACNA1 C expressed in HEK293 cells were observed by confocal fluorescence microscope;7.Cellular electrophysiological analysis and drug screening by whole cell patch clamp: The L-type calcium currents of HEK293 cells expressed with wide type and mutant CACNA1 C were recorded by whole cell patch clamp,the effect of testosterone(10 μM)in the current was detected.Isoproterenol(10 μM)and quinidine,the therapeutic drug of ER were also used to determine the influence to the calcium currents.The current-voltage(I-V)relationships,voltage dependent steady state activation(SSA)and steady state inactivation(SSI)were measured;8.Statistical analysis: All statistics were performed by using SPSS 19.0 software.Numerical data are presented as means ± standard errors of the mean(SEM).Statistical comparisons were performed with t-test or one-way ANOVA test.P values of <0.05 was considered to be statistically difference.P values of <0.01 was considered to be statistically significant difference.Results: 1.The mutation plasmid of CACNA1C-Q1916 R was successfully made,the 5747 th A-to-G mutation of CACNA1 C was confirmed by sequencing,and without any other mutations.2.The results of the immunohistochemical testing showed the protein expression level of Cav1.2 in the left ventricular of proband was lower than the healthy control(*P<0.05).The expression level of total m RNA(*P<0.01).and protein expression levels in total lysates,membrane and cytoplasmic(*P<0.05)of mutant Cav1.2 were lower than the wild type Cav1.2 heterologously expressed in the HEK 293 cells.3.The results of immunocytochemistry revealed normal trafficking ability of Cav1.2 mutant Cav1.2,but the global fluorescence intensity was weaker.4.The results of whole cell patch clamp presented a reduced current density in the mutant calcium channel compared to the wild calcium channel(*P<0.05,**P<0.01),there is no difference of the voltage dependent SSA and SSI between the 2 groups.The testosterone(10 μM)significantly reduces the current density(#P<0.05).The therapeutic drug of isoproterenol(10 μM)for ERS increase the wild calcium current density,there is no statistical significance of the increased mutant calcium current density,but the drug can left shift the voltage dependent SSA in both groups. The quinidine(5 μM)has no any effect on the calcium current density and voltage dependent SSA in the wild and mutant groups.Conclusions: 1.The CACNA1C-Q1916 R is a loss-of-function mutation.It causes the decreased m RNA and protein expression level of Cav1.2,and the reduced number of Cav1.2 in the membrane would be the cause of decreased calcium current,but has no effect on the protein location and trafficking.2.The CACNA1C-Q1916 R mutation is the main cause of SCD related to the early repolarization in the family we studied here.3.The testosterone exacerbates the decreased CACNA1C-Q1916 R mutant calcium current density.It confirmed that male gender is an important risk factor of SCD related to the early repolarization in the mutant carriers.4.There might be a putative therapeutic option of isoproterenol in the CACNA1C-Q1916 R mutant carriers with high risk of SCD related to the early repolarization in the family we studied here.