The Arrthymic Pathogenesis Of Gene Variants In KCNQ1 And SCN10A

Background:Inherited primary arrhythmic syndromes are a group of hereditary diseases with clinically rare but potentially predisposing to malignant arrhythmias and cardiac events.It includes long QT syndrome(LQTS),Brugada syndrome(Br S)etc.Cardiac ion channel malfunction by pathogenic gene mutations is currently regarded as the major cause of inherited primary arrhythmic syndromes.Mutation in a LQTS causative gene can be found in about 80%LQTS patients.LQTS type 1(LQT1)is the most common type of LQTS resulted from loss-of-function mutation in KCNQ1.About two third of Br S patients are still genetically undetermined.New gene variants,discovered by new methods such as next-generation sequencing,would provide comprehensive explanations in understanding cardiac electrophysiology.The important role of Nav1.8,encoding by SCN10A,in cardiac electrophysiology and arrhythmias like Br S and atrial fibrillation(AF)has been strongly suggested by multiple genome wide association studies.Gene variants in potential pathogenic genes aroused much concern in their associations with the pathogensis of arrhythmias.Objective:In this study,we aim to investigate the role of KCNQ1 mutation in LQT1 and SCN10A variants in Br S as well as the possible pathogenesis underlying early stage AF.We tend to explore the potential electrophysiological mechanisms of those identified possible pathogenic genes by patch-clamp and/or animal arrhythmic experiment(canine atrial rapid pacing models).Methods and Results:(1)KCNQ1 mutation in LQT1:A typical LQT1 patient(recurrent syncope and QTc495ms)with certain family history was identified and assessed clinically.Genome DNA was screened by whole-exon sequencing and a new mutation KCNQ1p.T312del was found.Detailed family and genetic investigations were conducted in the three-generation pedigree.This mutation presented autosomal dominant inheritance and complete penetrance in the evaluated LQT1 family.Family members(his mother and grandmother)carrying p.Thr312del displayed prolonged resting QT interval and recurrent syncope,especially during exercise.KCNQ1p.Thr312del caused significantly decreased Kv7.1(KCNQ1)and IKs(KCNQ1 and KCNE1)currents in transfected HEK293 cells(P<0.05,depolarizied potential from 0m V to 60m V);it also exhibited a slow activation process with a slower activation time constants in comparision to the wild-type IKs(P=0.036).In the mutant condition,IKs increased via protein kinase A activating but not during rapid pacing(2Hz),a significant suppression in IKs use-dependent facilitation.(2)SCN10A gene variants(Minor Allele Frequency/MAF>0.01)in Br S patients:Clinical and genetic analyses were performed in 179 enrolled Br S patients.Rs6795970 was strongly revealed to enhance the risk of Br S(OR=2.890,P=1.11×10^-24,the ESP database as the control);whereas rs7630989,rs57326399 and rs12632942 had the protective effect.Functional studies were carried out in SCN5A-SCN10A co-transfected cells.A positive shift(about+9m V,P=0.006)in steady-state inactivation and an accelerating slow recovery phase were noted in SCN10A-Val1073(A allele in rs6795970)transfected cells(slow phase time constants:A1073:38.47±10.88 ms vs.V1073:16.66±3.43 ms,P=0.029).(3)The pathogenic role of SCN10A in the early stage of AF:Both Nav1.5,the classical cardiac sodium channel encoding by SCN5A,and Nav1.8 were demonstrated in our study to be expressed in ganglionated plexi(GP)by immunohistochemical study.Sixteen canines were randomly administered either saline or Nav1.8 blocker,A-803467.Electrophysiological characteristics were compared between the two groups before and after 6-hour rapid atrial pacing(RAP).Compared with the control group,administration of A-803467 decreased the incidence of induced AF(87.5%vs.25%,P=0.015)and significantly suppressed the decrease in effective refractory period(ERP),caused by RAP in all recording sites.Patch clamp experiment was performed under 0.1?M A-803467 in SCN5A-SCN10A co-transfected TSA201 cells.Peak sodium current was reduced by 45.34%at-35 m V testing potential,and late sodium current by 68.57%at-20 m V testing potential.Besides,the steady-state inactivation curve was negative shifted about 12m V(P=0.007),Time constants in slow phase of recovery was significantly slower in the presence of A-803467(before and after the drug:20.51±5.61 ms vs.64.37±15.47ms,P=0.01).Pronounced use-dependent block were also observed after applying the drug in SCN10A-SCN5A contransfected cells.Conclusions:In conclusion,the role of gene variants(including mutation)in pathogenic genes,like KCNQ1 and SCN10A,is essential in the cardiac arrhythmic diseases(inherited primary arrhythmia syndromes).KCNQ1p.T312del,the new mutation detected in the present study,is responsible for LQT1.Both reduced ion conduction and gating character changes(slow activation time constants)underlie the pathogenic effect of this mutation.SCN10A variants can contribute the susceptibility to Br S.A allele in rs6795970(V1073)increase the risk of Br S.V1073 exhibited different gating characteristics in comparision to A1073 in transfected cells with a positive shift in steady-state activation curve and an accelerating time constants in the slow phase of recovery.Nav1.8 can exert its effect on acute AF through cardiac GPs.Nav1.8 inhibition though adminstating A-803467 at cardiac GPs attenutated the ERP shortening in the canine RAP model.A-803467 application significantly reduced the peak and late sodium current densities,accelerate slow phase of recovery and promote steady-state inactivation as well as use-dependent block in SCN5A-SCN10A co-transfected cells.SCN10A may interact with SCN5A via the influence of cardiac electrophysiological characteristics.