Associatioh Of Nodal And GDF1Gene Polymordhisms With Conaenital Heart Disease Susceptibility In Chinese Han Population And The Interaction Between Two Genes

Congenital heart disease (CHD) is the most common birth malformation and the leading cause of death in infants, occurrs in8out of1,000live births. To date, most of the CHDs (80%) have been considered to be the result of genetic and environment interactions, in which the genetic factor has been documented to be the most important deciding reason. NODAL signaling pathway is an important genetic pathway involved in cardiac asymmetric development. Sufficient evidences have shown its implications of genetic variations in the pathogenesis of CHDs. In this subject, we aimed to investigate the relationship between single nucleotide polymorphisms of two genes in NODAL signaling pathway, that is, Nodal and Growth/Differentiation factor1(GDF1), and CHD susceptibility in Chinese Han population based on previously reported NODAL pathway polymorphisms. The interactions between these two genes were also conducted.Part I. Association of Nodal gene polymorphisms with CHD susceptibility in Chinese Han populationAims:Nodal is the major ligand of NODAL signal pathway. Its proteolytic maturation and concentration are essential for the stability and range of NODAL signaling, and the determination of downstream gene expressions. It has been reported that two exon polymorphisms in Nodal gene can significantly reduce biological activity of Nodal. The present study was aimed to investigate whether two exon2polymorphisms of Nodal gene are associated with non-syndromic CHDs in Chinese Han population. Besides, two common variants in3’ untranslated region (3’ UTR) of Nodal gene were also selected for analysis in this study due to their possible roles of affecting gene expression in the post-transcriptional level.Methods:Genotype analyses using PCR-DHPLC (PCR-Denaturing High Performance Liquid Chromatography) and direct sequencing were performed on308non-syndromic CHD patients and314healthy controls for two exon SNPs (rs1904589and rs10999334) and two SNPs in3’ UTR (rs2279253and rs2279254) in Nodal gene.Results:The genotypes of all cases and controls show wild genotype GG for two exon SNPs, suggesting no polymorphic for the two exon locus in these study groups. And the overall genotype frequencies of two3’ UTR SNP have not significantly different between CHD patients and healthy controls. However, the rs2279253TT genotype have a weak association with conotruncal defects (CTDs)(P=0.029) after the case group was divided into two subgroups:CTDs and Non-conotruncal defects (Non-CTDs). Further stratified analysis for15subtypes of CHDs reveals that both rs2279253T and rs2279254G alleles are significantly associated with pulmonary valve atresia (PA) susceptibility (P=2.87×104,0.002). And after PA was divided into PA with an intact ventricular septum (PA+IVS) and PA with ventriucular septal defect (PA+VSD), rs2279253T shows association with the PA+IVS subtype (P=0.001). There is no significant correlation between the two SNPs and CHDs after stratified analysis by gender.Conclusions:Our study indicates that Nodal exon2polymorphisms may not involve in the pathogenesis of CHDs in Chinese Han population because they show no polymorphisms. But the SNPs within Nodal3’ UTR region may be implicated in the susceptibility and onset of some subtypes of CTDs in the Chinese Han population.Part Ⅱ. Association of GDF1gene polymorphisms with CHD susceptibility in Chinese Han populationAims: GDF1is a coligand of Nodal pathway heterodimerizing with Nodal. As an upstream gene, GDF1directly or indirectly regulates the expression of downstream gene in NODAL pathway and plays a key role in the asymmetric development of the heart. This study was aimed to investigate the association between GDF1gene polymorphisms and CHD susceptibility in Chinese Han population.Methods:Four tagSNPs of GDF1were selected by HapMap database and Haploview software, and were genotyped using PCR-based DHPLC and PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) in this cases-controls study.Results:The genotype and allele distributions of the four GDF1polymorphisms are all in Hardy-Weinberg equilibrium. The genotype and allele frequencies between CHDs and controls show no significant differences with any of the analyzed variants of GDF1. However, a weak statistical association exists between GDF1rs4808870and CTDs (P=0.027). Further stratified analysis for15subtypes of CHDs reveals that the SNP A allele is associated with PA susceptibility (P=0.003). The four SNPs of GDF1show strong linkage disequilibrium. Haplotype analysis reveals that TGGT is associated with decreased risk of CHDs, while CAGT appears to be a risk haplotype for CHD.Conclusion:Our results prove that genetic variations of GDF1may be associated with CHD risk, and these variations contribute at least in part to the development of some subtypes of CTD in Chinese Han population. Part Ⅲ. Interaction between Nodal and GDF1gene and the risk of CHD in Chinese Han populationAims:Congenital heart disease is a complex genetic disease, in which the intricate interactions between genes and genes are involved. Both Nodal and GDF1are Nodal ligands, their interactions may influence the regulation of NODAL signal for target gene expression. In this part, we aimed to investigate the interaction of the polymorphisms between these two genes and to construct an interaction model in Chinese Han population with CHDs.Methods:The interaction of Nodal and GDF1gene polymorphisms was analyzed by Multifactor Dimensionality Reduetion (MDR), and the Interaction Dendram and Interaction Graphs were furtherly used to confirm this interaction and construct the best interaction model.Results:The MDR analysis indicates that the best model for SNP-SNP interactions in GDF1gene is4sites model. It has highest cross-validation consistency (CVC) value10/10with61.00%testing accuracy. The MDR analysis in Nodal, GDF1gene shows that the best model is also4sites model (GDF1rs7250622, rs4808870, rs2075762and Nodal rs2279253), with8/10CV value and57.23%testing accuracy. The model that consists of GDF1rs7250622, rs4808867, rs2075762is the best one for CTDs groups. Interaction Graphs indicate significant synergistic effect between Nodal rs2279253and GDF1rs4808870, with0.98%Information gain (IG) value. While in Non-CTDs groups, the best model is composed of GDF1rs7250622, rs4808870, rs2075762and Nodal rs2279254. Interaction Graphs also show that there are no interactions in Nodal rs2279253, rs2279254and other SNPs.Conclusion:The study suggests a synergistic interaction in the four SNPs of GDF1gene, GDF1and Nodal gene in the pathologenesis of CHDs. The interaction between Nodal rs2279253with GDF1rs4808870may implicate in the pathogenesis of CTDs. The two polymorphisms of Nodal gene have independent effect on the susceptibility of Non-CTDs, but not dominant in the interaction effect.