The Molecular Mechanisms Study Of EVC Gene Variations In Ventricular Septal Defect

Background Congenital heart disease(CHD),a leading cause of non-infectious deaths in infancy,is one of the most common human birth defects that affects ~1% of live birth.As a complex disease,CHD is resulting from abnormal heart development.Heart development during the embryonic stage is regulated by a development network,including signaling pathways,transcription factors,and epigenetics.Gene mutations in any of these components may cause the development of CHD.The underlying molecular mechanisms for CHD remain largely unknown.Ventricular septal defect(VSD)is the most common type of CHD and accounts for ~30% of diagnoses.However,there have been few studies that focused on the molecular mechanism of VSD.Currently,there are several known pathogenetic genes of VSD,including GATA-binding protein 4(GATA4),GATA6,NK2 homeobox 5,T-box 1(TBX1)and TBX5.Ellis-van Creveld(EVC),an activator molecule of the Hedgehog(Hh)signaling pathway,is involved in embryonic development.Mutations in the EVC gene may lead to EVC syndrome.EVC syndrome is a rare autosomal recessive disorder characterized by a series of clinical manifestations including chondrodystrophy,postaxial polydactyly,ectodermal dysplasia,and ~60% of patients exhibit the CHD phenotype,of which,atrioventricular septal defect(AVSD),atrial septal defect and VSD are the most common.Previous studies on EVC gene mutations have focused on the EVC syndrome lineage and its influence on cartilage development.However,the EVC gene variations in patients with VSD and the potential underlying pathogenetic mechanisms have not yet been reported..Section 1 Genetic Analysis of EVC gene in the Patients with Ventricular Septal DefectObjective:To understand the EVC gene variations in Chinese Han patients with VSD and explore the genetic correlation between EVC gene variations and the development of VSD.Methods:1.All the VSD patients(n=65)and healthy controls(n=210)from the same ethnicity of Chinese Han population were recruited in this study.The direct DNA sequencing method was used to sequence the exons and exon-intron boundaries of the EVC genes to screen single nucleotide polymorphisms(SNPs)and mutation sites of EVC genes in clinical VSD probands and controls.Our study strictly complied with the medical ethical principles,and was approved by the Human Ethic Committee of the Second Affiliated Hospital of Nanchang University.The written informed consent was signed by the patients or their guardians prior to the present study.2.The variations of EVC were confirmed by the db SNP database(https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?gene Id=2121).Identification of SNPs or gene mutations was according to the gene frequency in the control group.The Polymorphism Phenotyping version 2(Poly Phen-2,http://genetics.bwh.harvard.edu/pph2),Sorting Intolerant from Tolerant(SIFT,http://sift.bii.astar.edu.sg/)and Mutation Taster(http://www.mutationtaster.org)programs were used to evaluate the disease-causing potential of detected EVC variants.Then the variant with highest correlation with disease was chosen for further research.3.Statistical analysis: statistical analysis was performed with the SPSS version20.0 statistical software.P<0.05 was considered to indicate a statistically signifcant difference.Genetic data were corrected for multiple comparisons using the Bonferroni correction test.The genetic balance of SNP genotypes was tested according to the Hardy-Weinberg equilibrium.Allele and genotype frequencies in the cases and controls were compared using chi-square tests.Risk prediction for VSD was expressed as odds ratio(OR)and 95% confdence interval(CI).Results:1.Through sequencing and screening of the EVC gene,a heterozygous nonsynonymous mutation was identified in a VSD patient.It was located in exon 3and was a C to G conversion at the 343 rd base of the coding region(c.343C>G),which caused a change from leucine to valine at the 115 th amino acid of EVC protein(p.L115V)and was not found in the other VSD patients and the healthy controls.2.Eleven EVC SNPs were detected in the patients with VSD,including c.221A>C(p.Q74P),c.772T>C(p.Y258H),c.969T>C(p.N323N),c.1026G>C(p.L342L),c.1068A>G(p.L356L),c.1320T>A(p.F440L),c.1333A>C(p.K445Q),c.1346C>A(p.T449K),c.1727G>A(p.R576Q),c.1854C>T(p.G618G),and c.2279G>A(p.R760Q).Four of the SNPs(c.1026G>C,c.1320T>A,c.1333A>C and c.1346C>A)were excluded for the association analysis owing to deviation from the Hardy-Weinberg equilibrium.The other seven SNPs were analyzed under five genetic models,including homozygous,heterozygous,dominant,recessive,and allele.The results revealed that the C allele of SNP c.221A>C was negatively associated with the risk of VSD development(OR=0.543,95%CI=0.300-0.981).The risk of VSD development in patients carrying the CC genotype of the c.1854C>T site was 2.246 times higher compared with the risk in patients carrying the TT genotype(95%CI=1.009-4.998).The C allele was the risk allele of VSD(OR=1.523,95%CI=1.019-2.278).Only the c.1727G>A site was significantly positively associated with the development of VSD following Bonferroni correction(p<0.007),indicating that the risk of VSD in people carrying the GA genotype was 4.411 times higher compared with the risk in people carrying the GG genotype(CI=2.047-9.506).In addition,in the dominant model,the risk of VSD in people carrying the GA or AA genotype was 4.2 times higher compared with people carrying the GG genotype(CI=1.982-8.901).The A allele in c.1727G>A was the risk allele of VSD(OR=2.039;CI=1.301-3.195).There were no significance differences in the distribution of the four other SNP sites in the five models.3.The SNP site c.1727G>A and the mutation site c.343C>G showed a probability of the pathogenicity variation in the PolyPhen-2 and MutationTaster software.The c.1727G>A site may cause an alteration in the splicing site and c.343C>G would be a pathogenic mutation.The Mutation Taster software showed that c.221A>C may lead to a change in the splicing site.Moreover,the c.1854C>T site was shown as a benign SNP.Conclusions:1.In this study,we firstly showed that the c.1727G>A polymorphism of EVC increased VSD susceptibility in a Chinese Han population.2.The c.343C> G may be a pathogenic mutation of the EVC gene.Section 2 Study on the Molecular Mechanisms of EVC-L115 V Mutation in Ventricular Septal DefectObjective:To investigate the effect of EVC-L115 V mutation on Hedgehog signaling pathway and its possible role in the development of VSD.Methods:The NIH3T3 cells were infected with recombinant adenovirus of EVC-WT-GFP and EVC-L115V-GFP respectively.The infected NIH3T3 cells treated with SAG,an Smo agonist of hedgehog signaling pathway,were divided into four groups,including EVC-WT,EVC-L115 V,EVC-WT+SAG and EVC-L115V+SAG.1.The effect of EVC-L115 V mutation on cell proliferation was examined.The cell proliferation marker Ki67 was used to observe the proliferation levels of NIH3T3 cells using immunofluorescence technique.The cells with Ki67-positive were visualized using a fluorescence microscope.The percentage of Ki67-positive cells was calculated per high power field.Then the effect of the EVC mutation on cell proliferation was further validated using an Ed U assay.The percentage of Ed U-labeled cells was calculated.Moreover,the protein level of cyclin D1 in all groups was detected by Western blot.2.The effect of EVC-L115 V mutation on cell apoptosis was observed.After the cells were treated with H2O2 to induce cell apoptosis,the protein levels of Bcl-2 and Bax were detected with Western blot and calculated as the ratio of Bcl-2/Bax,which is an indicator of the anti-apoptosis ability of cells.Furthermore,the percentage of apoptotic cells was assessed with TUNEL assay.3.The effect of EVC-L115 V mutation on the Hh signaling pathway was explored.The protein levels of Gli1 and Foxf1 a in NIH3T3 cells was detected by Western Blot.A Gli1 luciferase reporter gene plasmid was constructed.After Gli1 plasmid was transfected into cells using Lipofectamine LTX,the effect of EVC-L115 V mutation on the transcriptional activity of Gli1 was further evaluated by dual luciferase report gene system.Next,the m RNA level of Foxf1 a was examined by Real-time PCR.Furthermore,a Gli2-Flag plasmid was constructed and transfected into NIH3T3 cells to observe the levels of the two isoforms of Gli2(the suppressor form Gli2 R and the full-length form Gli2FUL)using western blot.In addition,co-immunoprecipitation technique was used to detect the effect of mutation on the binding between Smo and EVC.Results:1.Reduction of NIH3T3 cell proliferation by the EVC-L115 V mutation.Our results indicated that the percentage of Ki67-stained cells among the total cells in the EVC-L115 V group was significantly decreased compared with the WT group in the presence of the SAG(23±1.3% vs.30.4±1.8%,p<0.05).Also,the percentage of Ed U-labeled S-phase cells in the EVC-L115 V group was significantly decreased compared with that in the WT group in the presence of SAG(12.5±0.7% vs.15.6 ±0.4%,p<0.05).Furthermore,the western blot demonstrated that during the activation of the Hh pathway following SAG treatment,the expression of cyclin D1 in the EVC-L115 V group was significantly decreased compared with that in the WT group(p<0.05).2.Increase in apoptosis in NIH3T3 cells by the EVC-L115 V mutation.The western blot results showed that the protein levels of Bcl-2 and Bax in NIH3T3 cells did not change significantly between the EVC-L115 V group and the WT group in the presence of SAG.Interestingly,after the NIH3T3 cells were treated with H2O2 to induce cell apoptosis,the protein level of Bcl-2 was significantly decreased in EVC-L115 V mutant group,whereas the cells with EVC-L115 V mutant had an increase in the expression of Bax.The ratio of Bcl-2/ Bax protein level in cells with EVC-L115 V mutant was significantly lower than that in the WT cells.In addition,the TUNEL assay also indicated that NIH3T3 cells apoptosis level was also increased in EVC-L115 V mutant group compared with that in the WT group following H2O2exposure(15.0±0.7% vs.7.5±0.6%,p<0.01).3.Downregulation of Hh pathway activity by the EVC-L115 V mutation.The results demonstrated that,compared with the WT group,the m RNA levels of Foxf1 a,the protein levels and the transcription activity of the Gli1 protein were significantly decreased in the EVC-L115 V group when the Hh pathway was activated by SAG.In contrast,the protein level of Foxf1 a did not alter in both EVC-L115 V and WT groups.Moreover,compared with the WT group,it was shown that EVC-L115 V group had a significantly increase in the ratio of Gli2 R /Gli2 FUL and a decrease in the binding ability between EVC-L115 V and Smo.Conclusions:The molecular mechanism underlying the development of VSD induced by the EVC-L115 V mutation may be due to a reduction in the anti-apoptotic and proliferative abilities of cardiomyocytes via downregulation of Hh pathway activity.