Variants Of CITED2 Gene Promoter Region In Patients With Congenital Heart Disease And Cellular Function Verification

Objective: 1.To study the variant of CITED2 gene promoter region in three types of Chinese patients with congenital heart disease(CHD).2.To investigate whether the variants of CITED2 gene promoter region caused changes in cell function.It provides more evidence for genetic diagnosis of CHD and exploration of new therapeutic targets(including drug and gene therapy targets)..Methods: Analysis of CITED2 gene promoter region variants in DNA of three kinds of CHD patients of Han nationality in China(compared with normal human DNA and internationally used gene banks).A variety of cell function verifications were performed on the found variants,and bioinformatics analysis was used to study the effects of these variants on transcriptional activity to reveal their role in the pathogenesis of CHD.The specific operation process is as follows :1.The peripheral blood DNA samples of CHD patients and healthy people were collected retrospectively from the sample bank.The basic clinical data of the two groups of target population were collected according to the inclusion and exclusion criteria,and all samples of peripheral blood and DNA extraction were performed.2.The primers of CITED2 gene promoter sequence were designed,and PCR amplification and Sanger sequencing were performed on about-2000 bp upstream of the transcription start point of CITED2 gene promoter region in the experimental group and the control group to find abnormal variant sites and gene polymorphism base sites.3.To study the effect of CITED2 gene promoter variant on its activity: the wild-type and variant promoter fragments of CITED2 gene were inserted into the luciferase reporter gene vector(p GL3-Basic)to construct CITED2 gene promoter-variant recombinant plasmid and CITED2 gene promoter-wild-type recombinant plasmid.The two recombinant plasmids and internal reference plasmid were cotransfected into mouse cardiomyocytes(HL-1)/ Human embryonic kidney cells(HEK-293),cultured for 48 hours.The dual luciferase reporter assay system was used to detect the regulatory effect of the variant and wild-type CITED2 gene promoter fragment on the downstream reporter gene.The experiment was repeated at least three times.4.Designing probes for wild-type and target genes with sequence mutation sites,probes were designed to verify whether the binding ability of transcription factor(TF)to promoters changed in electrophoretic mobility shift assay(EMSA).In the JASPAR database,we explored whether variants in the gene promoter region affected the potential transcription factor binding site(TFBS)of the CITED2 gene.5.The experimental results were statistically analyzed to demonstrate the role of CITED2 gene promoter variants in the pathogenesis of CHD.The results of CITED2 gene promoter sequencing and functional verification were sorted out to explore the relationship between CITED2 gene promoter region variant and CHD.Results: 1.Based on inclusion and exclusion criteria,for a total of 1290 blood samples were collected from Chinese Han population who met the criteria of this study.There were 332 patients with ASD,312 patients with TOF,353 patients with PDA and 293 normal controls.2.A total of 15 variants were found after PCR amplification and sequencing,and 12 variants were only found in the CHD group.The other three common variants[g.4285T>G(rs12333191),g.4357G>A(rs76757432),and g.5122C>A(rs570422697)]were excluded from further studies.(1)Four types of variants were found in ASD group: g.4078A>C(rs1165649373),g.4240 C>A(rs1235857801),g.4935C>T(rs111470468),and g.5027C>T(rs112831934);(2)Six types of variants were found in TOF group: g.4165C>T,g.4660T>G(rs375786932),g.4698A>G(rs529363037),g.4935C>T(rs111470468),g.5108C>T(rs995827210),and g.5027C>T(rs112831934).(3)Seven types of variants were found in TOF group: g.3949C>T(rs76315931),g.4047＿4048ins C(rs1223604040),g.4330＿4331ins C(rs1298132522),g.4808＿4815del GGGGCGAC(rs983891177),g.4935C>T(rs111470468),g.5108C>T(rs995827210),and g.5027C>T(rs112831934).3.Construction of CITED2 gene promoter p GL3-Basic dual fluorescence reporter gene vector.They were p GL3-WT(wild type),p GL3-4078 C,p GL3-4240 A,p GL3-4935 T,p GL3-5027 T,p GL3-4165 T,p GL3-4660 G,p GL3-4698 G,p GL3-5108 T,p GL3-3949 T,p GL3-4047＿4048ins C,p GL3-4330＿4331ins C,p GL3-4808＿4815del GGGGCGAC.After sequencing,the insertion variant sequence was correct and the recombinant plasmid was successfully constructed.4.After transiently transfected into cells by liposome,it was found that 12 variants found in CHD group could significantly affect the transcriptional activity of CITED2 gene promoter by dual luciferase reporter analysis after 48 hours,and the differences were statistically significant(p<0.01 or 0.05).5.EMSA experiments were performed using wild-type or variant biotin-labeled probes.The experimental results showed that the variants of the promoter may indeed affect the transcription by affecting the binding ability of the CITED2 gene promoter to TF.The prediction results of the JASPAR database showed that variants in the CITED2 promoter region may destroy or produce potential TFBS and affect CITED2’s involvement in related TFs and pathways to regulate normal heart development.Conclusion: This study found that there are variants in the CITED2 gene promoter region in the three major diseases(ASD,TOF,PDA)of CHD in Chinese.A total of 12 variants were found,of which 1 were reported for the first time.Through cell function verification experiments and bioinformatics analysis,it was found that these 12 variants affected the expression of CITED2 protein and the binding of related TFs.Therefore,these variations play an important role in the pathogenesis of CHD.This work deepens the study of the molecular mechanism of CHD,and provides a new scientific basis for the development of new diagnostic and therapeutic targets and drug interventions in the diagnosis and treatment of CHD in the future.