Identification Of Mutations Of CITED2 And ISL1 Gene Promoter And Functional Verification In Isolated Ventricular Septal Defects

BackgroundCongenital heart disease(CHD)is a congenital malformation that seriously threatens the lives and health of children,and its incidence ranks first among patients with congenital birth defects.Besides,CHD is the most frequent fetal losses,accounting for the proportion of 30%to 50%.Ventricular septal defect(VSD)is the most common CHD,comprising approximately 20%to 35%of CHD.It is increasingly recognized that genetic factors play a crucial role in the etiology of VSD.However,despite great improvements in genomic technologies,little progress has been made towards understanding the mechanism of the formation of VSD.A large amount of studies indicates that mutations in the coding regions of heart-related genes,such as CITED2 and ISL1,may be involved in the formation of VSD.and evolution of VSD.Nevertheless,there is currently no research to explore the mutations in the promoter regions of CITED2 and ISL1 in VSD patients and to investigate the relationship between the mutations and the development of VSD.Therefore,this research aims to explore mutations in CITED2 and ISL1 gene promoter regions in VSD patients,as well as their impacts on the development of VSD,to provide a theoretical foundation for the genetic diagnosis and precise treatment of VSD and to improve the prognosis of patients.ObjectivesThe aim of this study is to investigate the role of mutations in CITED2 and ISL1 gene promoters on the development of VSD and provide new insights into the etiology of VSD.MethodsThis study is designed to screen VSD patients who underwent surgery from January 2018 to August 2019 in our hospital.After matching them with healthy children by gender and age,they were set as the case group and the control group respectively.And then,we collected blood samples of all subjects,extracted genomic DNA,and performed target gene fragments amplification and Sanger sequencing.We investigated the CITED2 and ISL1 gene promoter mutations and performed cellular functional experiments by using the dual-luciferase reporter gene assay to verify its impacts on gene expression.Besides,by means of electrophoretic mobility shift assay(EMSA)and JASPAR database,we explored the putative binding sites for transcription factors that might be disrupted,created,or affected by gene promoter mutations.ResultsAfter age and gender matching,there were 400 subjects enrolled in this study,including 200 isolated and sporadic VSD patients and 200 healthy children.A total of 5 mutations were identified only in the VSD group by Sanger sequencing in the CITED2 gene promoter region.Using dual-luciferase reporter assay,we found four of these 5 mutations identified significantly decreased the transcriptional activity of the CITED2 gene promoter(P<0.05).Further,EMSA experiments revealed that three mutations significantly affected the binding affinity of transcription factors.Bioinformatic analysis with the JASPAR databases found that a cluster of putative binding sites for transcription factors was disrupted,created,or altered by these mutations,leading to low expression of CITED2 protein and development of VSD.In the ISL1 gene promoter region,5 mutations were found only in VSD patients by Sanger sequencing.Cellular functional experiments demonstrated that three mutations significantly decreased the transcriptional activity of the ISL1 gene promoter(P<0.05).EMSA experiments revealed that these three mutations affected the binding affinity of transcription factors to the ISL1 gene promoter.Further analysis with the online JASPAR database,a cluster of putative binding sites for transcription factors that may be disrupted,created,or altered by these mutations were predicted,resulting in the low expression of ISL1 protein and the formation of VSD.ConclusionsOur study has for the first time identified mutations in the CITED2 and ISL1 gene promoter region in the Han Chinese patients with isolated and sporadic VSD.Additionally,these mutations significantly alter the expression of the CITED2 and ISL1 gene and affect the binding of transcription factors that may lead to the development of VSD.This study may provide new insights into the mechanism of the formation of VSD.