Identification And Functional Verification Of Pathogenic Genes In Familial Atrial Fibrillation

Background:Atrial fibrillation(AF)is the most common cardiac arrhythmia.The heredity of atrial fibrillation has been extensively studied since the first report of familial atrial fibrillation(FAF)in 1936.There is a genetic susceptibility for FAF.These mutations have been detected in ion channel and non-ion channel genes.Gene research on FAF would contribute to better understanding the molecular biological basis of FAF,and making effective treatment decisions in FAF patient management.It will be critical to develop specific small-molecule drugs for FAF gene therapy and the pathophysiological mechanisms of different channels.Objective : This study focused on the suspected pathogenic genes of atrial fibrillation in a familial atrial fibrillation family,as well as the mutation sites and electrophysiological experiments at the cellular level.Investigating the effect of suspected pathogenic gene mutation sites on current cell changes in order to understand the pathogenic mechanism of the aforementioned mutations.Methods:The gene exon library was obtained through the whole exon gene testing of the proband and the 6 lineal family members that could be collected.Then,based on this gene library,SNP bioinformatics was used to analyze the mutated genes common to AF patients but not non-existing in AF patients,and to screen out the suspected pathogenic genes related to the occurrence of AF.The above analysis was performed on the probands and their 8lineal family members by Sanger method,and the fragments before and after the suspected pathogenic gene mutation sites were screened out.Sequencing analysis and first generation validation were performed to identify the mutant gene and its mutation site.And compared with the same gene mutation sites of other AF members in the family,to further identify the pathogenic mutation sites.Then,the above-mentioned pathogenic mutation sites were transfected into Cos7 cells,and the patch clamp technique was used to record the change of ion channel currents.To explore the influence of the suspected pathogenic gene mutation sites on the changes of ion current in cardiomyocytes,so as to understand the pathogenesis of AF.Resuits: According to the whole exon gene detection and preliminary screening of probands and their 6 lineal family members,a total of 7 suspected pathogenic genes of af were found,namely KCNQ1(c.625T>C)、 FAM160A2(c.1375C>T)、MUC5B(c.12272C>T)、PRAMEF1(c.652C>T)、NEB(c.9052G>A)、NR4A2(c.9052G>A)、KIAA1755(c.1898C>T)。We further verified by Sanger method that only 4 suspected pathogenic genes were KCNQ1(c.625T>C)、FAM160A2(c.1375C>T)、MUC5B(c.12272C>T)、PRAMEF1(c.652C>T).Three suspected pathogenic genes KCNQ1(c.625T>C)、FAM160A2(c.1375C>T)、 MUC5B(c.12272C>T)were identified by comparison with the mutation loci of other members of the family.The mutant target genes were transfected into Cos7 cells.Patch clamp technique showed that compared with wild-type KCNQ1-KCNE1 cells,the instantaneous outward potassium current of KCNQ1 mutant co-transfected with KCNE1 cells was significantly reduced.But other genes have no obvious effect on the change of outward current of potassium ion.Conclusion:1.We found heterozygous T>C missense mutation(C.625 T >C)in the exon of chromosome 11 of KCNQ1 in a family with familial atrial fibrillation.Heterozygous C>T missense mutation(C.1375 C >T)on the exon of chromosome 11 of FAM160A2 and newly discovered heterozygous C>T missense mutation(C.12272 C >T)on the exon of chromosome 11 of MUC5 B.2.KCNQ1(C.625T>C)was a functional loss mutation,while FAM160A2(C.1375C>T)and MUC5B(C.12272C>T)did not significantly affect the outward current of potassium ions.