Mutation Screening And Functional Analysis Of NOTCH1 Gene In Tetralogy Of Fallot

Congenital heart disease (CHD) is the most common birth defect in the world, nearly 1-2% infants worldwide suffer from this disease. CHD is the main reason to the death of infants under 1 year old. Although CHD is a familial disease which indicating that hereditary factors have a certain impact on the generation of heart malformations, the majority of CHD does not follow the Mendel’s laws of inheritance. CHD is often accompanied by some other developmental malformation. The common symptoms includes atrial septal defect, ventricular septal defect, Tetralogy of Fallot and bicuspid aortic valve, etc. The disease has seriously affected the quality of China’s population, posing a serious burden to families and society. Currently, in many studies, the relationship between genes and CHD can be discovered by determination of polymorphic and mutation detection for the coding region and non-coding regulatory region of a particular gene, which can establish the theoretical foundation for the molecular diagnosis of such complex diseases.Notch signaling pathway is an important and evolutionarily conserved regulatory pathway in heart development. It plays different roles in myocardium and endocardium. Through the interaction of various myocardial and neural crest signaling pathway, Notch signaling pathway regulates the morphogenesis of heart chamber; in addition, Notch can also regulate the amplification and differentiation of myocardial cells, and participates in the specialization of coronary vessels. Many studies indicated that abnormality of NOTCH1, JAG1 genes in this pathway was related to the occurrence of CHD. In this article, by the case-control approach, rare mutation screening and functional analysis are carried out for NOTCH1 gene coding region in the samples of TOF. Among the case of 69 patients,6 missense mutations (c.578G>A, C.2501C>T, c.4507G>A, c.6229G>C, c.6853G>A, c.7366A>G) were found in the Notch 1 extracellular domain (NECD) and Notch1 intracellular domain (NICD). These 6 missense mutations corresponds to G193D, A834V, G1503S, A2077P, V2285I, I2456V respectively, which were all located in the important structural domains of Notchl protein. We carried out functional analysis for the three mutations in NICD (A2077P, V2285I, I2456V). The results of dual luciferase reporter system experiments indicated that A2077P affected the function of NICD that regulating the transcription of downstream target genes, which greatly reduced the transcription function of HEY2 and SNAIL2 genes; V2285I also showed adjustive function for the transcription of HEY2 gene; but the influence caused by the mutation of I2456V was not significant. The results of nuclear localization experiments showed A2077P and V2285I affected the normal behavior of NICD protein that entering nucleus, which led to the NICD retain on the cell membrane and outside the karyotheca but not in the nucleolus; and the influence caused by mutation of I2456V was also not significant.Folic acid metabolic pathway is a complex metabolic pathway which uses folic acid as an important carrier of one-carbon units. It connects the epigenetics and the synthesis and repair of DNA. Some literatures recorded that disorder of folic acid metabolism was related to many complex diseases, and it is the potential risk factor of the generation of CHD. In folic acid pathway, both Vitamin B12 (VB12) and homocysteine (Hey) are important intermediates, and the concentration levels of them directly affect the stability of the entire folate metabolism. According to the results of genome-wide association analysis, we screened 6 polymorphic sites which directly affect the concentration of VB12 in human bodies to carry out association analysis for our case-control samples of CHD. Results showed that the The variant rs 11254363 of CUBN gene was associated with decreased risk of developing CHD in both the separate and combined case-control studies. Although no obvious relatedness showed between other five polymorphic sites (rs602662, rs601338, rs492602, rsl 801222, rs526934) and CHD, according to the results of GWAS, we could not exclude the role played by the FUT2, CUBN, TCN1, genes with the risk of CHD.This study explored the genetic mechanisms of CHD from the different perspective through Notch signaling pathway and folic acid metabolic pathway, it verified the function of being a transcription factor caused by the mutation of NOTCH1 gene, which produces the phenotype of Tetralogy of Fallot; and several candidate genes and polymorphic sites related to CHD were found from GWAS results by using a novel idea.