The Mechanistic Exploration On The Role Of NPPA And Corin In Congenital Ventricular Septal Defect

OBJECTIVE:Congenital heart defect(CHD)is the most common type of congenital malformations.It accounts for about 28%of all congenital malformations.It is the leading cause of noninflammatory death in infants.Ventricular septal defect(VSD)is the most common congenital heart disease,and the incidence rate is about 2-6/1000.Genetic factors play an important role in the pathogenesis of VSD.Except single-gene mutations or chromosomal aberrations,most VSDs are sporadic and unexplained.In recent years,with the development of next-generation sequencing techniques,more and more new genetic factors are found to be related to the occurrence of CHD.In this study,we hope to explore the VSD candidate gene of Chinese Han population by exon sequencing with large sample size.Then we clarify the pathogenicity of the gene and its pathogenic molecular mechanism in cell and experimental animal model.METHODS:We first performed exon sequencing of 32 members(including 14 VSD patients and 18 normal members)of 8 VSD families,screened out de novo missense mutations in each family.Then 192 VSD patients and 96 normal controls were subjected to targeted sequencing in a case-control study,and the mutated genes in VSD patients were searched.After NPPA was selected as candidate gene,841 VSD patients was treated with NPPA targetted sequencing and compared with NHLBI-ESP database.HEK293T cells were used to study the effect of mutant p.M51T and p.R126Q on NPPA.The effects of NPPA and Corin on HL-1 cells were also studied.The effect of depressed NPPA and Corin on cell apoptosis and migration was investigated by HL-1 cell.Animal experiment was to verify Corin knockout of mouse embryonic heart phenotypes.RESULTS:For exon sequenceing with eight VSD families,we found 10 de novo missense mutations and 5 de novo synonymous mutations.Of 10 de novo missense mutations,only NPPA and TBC1D3G expression were decreased in VSD heart tissue,and NPPA was mainly expressed in cardiac tissue.For target sequencing,compared with the 96 Han population,and 20 genes were enriched in 192 VSD cases.NPPA was enriched in VSD patients(P=0.0007)and included in the pathway for cardiogenesis(BMP→ SMAD→ATF2→NPPA).Then we targeted NPPA gene by sequencing 841 cases of VSD.Compared with the 6500 NHLBI-ESP database,the rare missense mutations on NPPA were significantly enriched in VSD patients(P=0.0009).A total of 7 rare missense mutations in NPPA were found in VSD patients.Mutation p.M51T can reduce the NPPA mRNA and protein level due to mRNA degradation acceleration.Corin can cleave NPPA into NT-proANP and ANP.Mutation p.R126Q can significantly reduce the cleavage effect of Corin on NPPA.NPPA and Corin can be expressed on the ventricular septum of embryo period,and the expression position is the same.Flow cytometry showed that downregulation of NPPA and Corin could affect the apoptosis of HL-1 cell.Transwell experiments showed that down-regulation of NPPA and Corin could reduce the migration of HL-1.Coparmed with WT mice,VSD close dalay in Corin knockout mice embryo,and the closure time window is E16.5 day.CONCLUSION:Through exon sequencing and target sequneicng,NPPA was screened out as a susceptibility gene for VSD in Chinese Han population.The mutant p.R126Q on NPPA gene can significantly reduce the effect of Corin on NPPA cleavage.Down-regulate NPPA and Corin can affect the apoptosis and reduce the ability of cardiomyocytes to migrate.Corin knockout mouse embryonic heart showed VSD phenotype on E14.5 and E15.5.In this study,the pathogenicity of NPPA and Corin gene was clarified by the study of large sample size sequencing,cell function and experimental animal model,so the molecular mechanism of NPPA and Corin gene were elucidated.It was of great theoretical and practical value for gene diagnosis and new drug research for VSD in the future.