Correlation Study Of NKX2.5 Gene Mutation And Atrial Septal Defect

Objectives:Congenital Heart Disease(CHD)is one of the most common congenital defects in neonates,and also one of the main causes of infant death.Genetic factors are recognized to be closely related to CHD worldwide.In recent years,the incidence of CHD has been increasing year by year.In order to clarify the relationship between CHD and genetic factors,this study used whole exon sequencing and bioinformatics analysis on collected clinical blood samples to further find CHDrelated susceptibility genes and mutation sites,and conducted functional verification at the cellular level.Methods:Part I: Three CHD families were screened from the biological information sample database of the First People’s Hospital of Yunnan Province,and clinical sample data of the proband and their first-degree and second-degree relatives were collected.DNA extraction and whole exon sequencing were performed on the proband and their families to screen out suspected pathogenic mutations,which were verified by bioinformatics.False positive pathogenic mutations were eliminated by Sanger sequencing.The identified suspected pathogenic mutations were validated on clinical samples.Part II: According to the three suspected pathogenic mutations of NKX2.5 gene screened out in the first part,the point mutation plasmid was constructed.The m RNA level of NKX2-5 was detected by q PCR after transfection to confirm the success of transfection.WB and cell scratch tests were used to explore the effect of NKX2.5 gene point mutation on the function of human cardiomyocytes,and to further clarify the relationship between NKX2.5 gene and CHD.Results:Part I: DNA extraction and WES were performed from a total of 10 family members in three lineages to screen for three NKX2.5 gene mutation loci that have not been reported at home or abroad: NNXK2.5 c.1265G>T,NXK2.5 c.1433 A>G,and NXK2.5 c.1500 G>C.After Sanger sequencing to verify that NKX2.5 was excluded After Sanger sequencing to rule out false positive NKX2.5 point mutations,clinical sample validation analysis revealed that NXK2.5 c.1265G>T,NXK2.5 c.1433 A>G,and NXK2.5 c.1500 G>C mutations were closely associated with the development of CHD.Part II: According to the three point mutation results of NXK2.5c.1265G>T,NXK2.5c.1433 A >G,NXK2.5c.1500 G>C,the point mutation plasmid was successfully constructed.The results of WB and Elisa showed that c Tn I expression in cardiomyocytes was significantly increased after transfer to the point mutation plasmid,which indicated that the point mutation may cause myocardial cell damage.Conclusions:1.In this study,three unreported point mutation sites of NKX2.5 gene were found:NXK2.5c.1265G>T,NXK2.5c.1433 A>G,and NXK2.5c.1500 G>C.2.The mutations of the NKX2.5 gene may be a major factor in atrial septal defects during early heart development.