Nodal、GDF1基因多态性与中国汉族先天性心脏病的关联研究及基因间的交互作用研究

发布时间：2018-05-29 18:52

本文选题：先天性心脏病 + NODAL信号通路　；参考：《兰州大学》2012年博士论文

【摘要】：先天性心脏病(Congenital heart disease,CHD)是最常见的先天缺陷性疾病,也是引起婴幼儿死亡最常见的原因,其总体发病率约8‰。目前,大多数(80%)先心病被认为是由遗传和环境等因素共同作用导致的复杂疾病,其中遗传因素对先心病的发生起决定性作用。NODAL信号通路是参与心脏不对称发育的重要通路之一,有很多证据表明NODAL通路基因变异与先心病发病有关。本文就国际先心病研究中所发现的NODAL通路多态性问题展开讨论,首次探讨NODAL通路中重要的两个基因Nodal和生长/分化因子(Growth/Differentiation factor1, GDF1)多态性与中国先天性心脏病的相关性问题,并以此进一步分析先心病中的Nodal基因与GDF1基因间的交互作用。第一部分Nodal基因多态性与中国汉族先天性心脏病的关联研究目的：Nodal是NODAL信号通路的主要配体,Nodal配体对维持NODAL信号的稳定性和作用范围、保证下游基因的正常表达起着非常重要的作用。研究发现,Nodal基因的两个单核苷酸多态性能够显著降低Nodal的生物学活性。本研究旨在探讨Nodal基因这两个第二外显子区单核苷酸多态性是否与中国汉族单纯性先天性心脏病患者的发病相关,同时也对与转录后基因表达水平调节有关的3'UTR区的两个多态位点进行了相关性分析。方法：采用PCR-变性高效液相色谱(PCR-Denaturing High Performance Liquid Chromatography, PCR-DHPLC)和直接测序的方法对308例先心病患者和314例健康对照进行了Nodal基因第二外显子区rs1904589和rs10999334及3'UTR区rs2279253和rs2279254的病例-对照关联研究。结果：Nodal的2个外显子SNPs位点在中国汉族病例组和对照组中均无多态性,全部表现为野生基因型GG；Nodal基因3'UTR的两个多态性位点rs2279253和rs2279254虽与先心病总体并无相关性,但将先心病分为圆锥动脉干畸形组(conotruncal defects, CTDs)和非圆锥动脉干畸形组(Non-conotruncal defects, Non-CTDs)西大类后分层分析,rs2279253TT基因型与CTDs发病风险有潜在关联性(P=0.029)；随后在15种先心病亚型中进行的分析表明,rs2279253T和rs2279254G等位基因频率都与CTDs组中肺动脉闭锁(pulmonary atresia, PA)显著相关(P=2.87×10-4,0.002)。肺动脉闭锁又可以分为伴有室间隔缺损型(pulmonary atresia with ventriucular septal defect, PA+VSD)和室间隔完整型(pulmonary atresia with an intact ventricular septum, PA+IVS)两种类型。经统计发现,rs2279253位点次等位基因与PA+IVS显著相关(P=0.001)。按性别分层分析表明两个位点与先心病无明显相关性。结论：我们的研究提示中国汉族群体Nodal基因第二外显子区无多态性,参与中国汉族单纯性先心病发病的可能性很小,但Nodal3'UTR区域的单核苷酸多态性,可能与中国汉族人群中CTDs的某些亚型的易感性有关。第二部分GDF1基因多态性与中国汉族先天性心脏病的关联研究目的：GDF1也是NODAL信号的配体,可与Nodal通过异源二聚作用形成共配体。作为NODAL信号通路的上游基因,GDF1直接或间接调控通路中下游基因的表达,在心脏左右不对称发育中发挥关键作用。本研究旨在探讨GDF1基因多态性是否与中国汉族先天性心脏病(CHD)患者的发病相关。方法：利用HapMap和Haploview筛选出GDF1四个tagSNP (rs7250622, rs4808867,rs4808870, rs2075762),采用PCR-DHPLC和PCR-限制性片段长度多态性(PCR-Restriction Fragment Length Polymorphism, PCR-RFLP)的方法对这四个tagSNP进行先心病病例-对照关联研究。结果：所检测的GDF1的四个tagSNPs位点在病例组和对照组的基因型频率分布均符合哈迪-温伯格平衡。这四个tagSNP在等位基因频率和基因型的分布上与中国人群总体先天性心脏病的易感性均无相关性；但是,按是否与圆锥动脉干发育有关对病例进行分层后发现,rs4808870位点AA基因型与CTDs组有关(P=0.027),进一步在先心病亚型中的分析也表明GDF1rs4808870的A等位基因与肺动脉闭锁相关(P=0.003)；对GDF1基因4个多态性位点的连锁不平衡分析中发现,4个位点呈较强连锁不平衡；单倍型TGGT在中国人群中起保护性作用而CAGT是先心病发生的危险单体型。结论：GDF1基因多态性可能与中国汉族先天性心脏病(CHD),尤其是CTDs某些亚型的易感性有关。第三部分中国先天性心脏病Nodal、GDF1基因的交互作用研究目的：先天性心脏病属于复杂性遗传病,其发病涉及到基因与基因之间错综复杂的相互作用。Nodal和GDF1同属于NODAL信号通路配体,两者能够相互作用,影响NODAL信号对靶基因表达的调控。本研究探讨了这两种基因多态性在中国汉族先天性心脏病中的交互作用及其作用的模式。方法：利用多因子降维法(Multifactor Dimensionality Reduction,MDR)分析Nodal和GDF1基因间的交互作用；进一步应用互作系统树及互作图谱来分析确证SNP的基因与基因之间的相互作用关系及最佳互作模型。结果：GDF1四个多态位点间的互作分析结果显示,由4个多态位点共同组成的模型为相互作用的最佳模型,不仅P0.0001、十重交叉验证CVC的一致性最大(10/10),而且测试精确度最大(61.00%)；对Nodal与GDF1之间的交互作用分析发现,由GDF1rs7250622、rs4808870、rs2075762和Nodal rs2279253组成的4因子为最佳互作的模型,CVC值为8/10,测试精确度为57.23%；在圆锥动脉干畸形组中由GDF1rs7250622、rs4808867、rs2075762组成的3因子为最佳互作的模型,互作图谱表明Nodal rs2279253与rs4808870有显著的协同效应,互作效应IG墒为0.98%；而在非圆锥动脉干畸形组,由GDF1rs7250622、 rs4808870、rs2075762和Nodal rs2279254组成的4因子为最佳互作的模型,互作图谱分析结果显示Nodal rs2279253和rs2279254之间及与其他大多数位点均无互作效应。结论：GDF1四个多态位点间有显著的协同效应；先心病中GDF1基因与Nodal基因间存在一定的协同作用；Nodal rs2279253与GDF1rs4808870之间的协同作用对圆锥动脉干畸形的发生可能产生一定的影响；Nodal两个多态位点在非圆锥动脉干畸形的发病中单独起作用,在互作效应中并不占主导地位。
[Abstract]:Congenital heart disease (CHD) is the most common congenital defect. It is also the most common cause of infant death. Its overall incidence is about 8 per thousand. At present, most (80%) congenital heart disease is considered to be a complex disease caused by the combination of genetic and environmental factors, of which hereditary factors are associated with congenital heart disease. .NODAL signaling pathway is one of the important pathways involved in the asymmetric development of the heart. There is a lot of evidence that the genetic variation in the NODAL pathway is related to the onset of congenital heart disease. This paper discusses the polymorphism of NODAL pathway in the study of the international congenital heart disease, and discusses the two important genes in the NODAL pathway for the first time, Nodal and birth. The relationship between the Growth/Differentiation factor1 (GDF1) polymorphism and Chinese congenital heart disease (CCHS), and to further analyze the interaction between the Nodal gene and the GDF1 gene in the congenital heart disease.
Part one association between Nodal gene polymorphism and congenital heart disease in Chinese Han population
Objective: Nodal is the main ligand for the NODAL signaling pathway. The Nodal ligand plays a very important role in maintaining the stability and scope of the NODAL signal and ensuring the normal expression of the downstream genes. It is found that the two single nucleotide polymorphisms of the Nodal gene can significantly reduce the biological activity of Nodal. This study aims to explore the Nodal gene. Whether the single nucleotide polymorphisms of these two exons are related to the incidence of simple congenital heart disease in Chinese Han people, and the correlation analysis of two polymorphic loci related to the regulation of the post transcriptional gene expression level in 3'UTR region.
Methods: the PCR- denaturing high performance liquid chromatography (PCR-Denaturing High Performance Liquid Chromatography, PCR-DHPLC) and direct sequencing were used to study 308 cases of congenital heart disease and 314 healthy controls. The case control association of rs1904589 and rs10999334 and 3'UTR region rs2279253 of Nodal gene was carried out in 314 cases of healthy controls.
Results: the SNPs loci of 2 exons of Nodal were not polymorphic in the Han Chinese case group and the control group. All of them were wild genotypic GG. The two polymorphic loci of Nodal 3'UTR, rs2279253 and rs2279254, were not related to the congenital heart disease, but the congenital heart disease was divided into the conical artery dry deformity group (conotruncal defects, CTDs). The Non-conotruncal defects (Non-CTDs) group (defects, Non-CTDs) has a potential correlation with the risk of CTDs (P=0.029). Subsequently, the analysis of the 15 subtypes of congenital heart disease showed that the frequencies of rs2279253T and rs2279254G alleles were all with the pulmonary atresia in the CTDs group (pulmonary at. Resia, PA) is significantly correlated (P=2.87 x 10-4,0.002). Pulmonary atresia can be divided into two types with ventricular septal defect (pulmonary atresia with ventriucular septal defect, PA+VSD) and ventricular septal integrity. A significant correlation was found between PA+IVS and P=0.001 (P=0.001). According to gender stratification analysis, there was no significant correlation between the two loci and congenital heart disease.
Conclusion: our study suggests that there is no polymorphism in the Nodal gene of the Han population in Chinese Han population. The possibility of participating in the onset of simple congenital heart disease in Chinese Han nationality is very small, but the single nucleotide polymorphisms in the Nodal3'UTR region may be related to the susceptibility of some subtypes of CTDs in Chinese Han population.
The second part is the association between GDF1 gene polymorphism and congenital heart disease in Chinese Han nationality.
Objective: GDF1 is also a ligand of NODAL signal, which can form CO ligand with Nodal through heterogenous two polymerization. As the upstream gene of NODAL signaling pathway, GDF1 directly or indirectly regulates the expression of the middle and downstream genes in the pathway and plays a key role in the asymmetric development of left and right heart. The purpose of this study is to explore whether the polymorphism of GDF1 gene is associated with the Han nationality in China. The incidence of congenital heart disease (CHD) is related.
Methods: GDF1 four tagSNP (rs7250622, rs4808867, rs4808870, rs2075762) were screened by HapMap and Haploview, and PCR-DHPLC and PCR- restrictive fragment length polymorphism (PCR-Restriction Fragment) was used to carry out a case-control study of the four cases of congenital heart disease.
Results: the genotypic frequency distribution of the four tagSNPs loci of the GDF1 in both the case group and the control group was consistent with Hardy Weinberg balance. These four tagSNP were not related to the susceptibility of the Chinese population to the overall congenital heart disease in the allele frequency and genotype distribution; however, whether it was developed with the stem of the conical artery or not After stratifying the cases, the rs4808870 loci AA genotype was associated with the CTDs group (P=0.027). Further analysis in the subtype of congenital heart disease showed that the A allele of GDF1rs4808870 was associated with pulmonary atresia (P=0.003), and the linkage disequilibrium analysis of the 4 polymorphic loci of the GDF1 gene showed that the 4 loci were strongly linked to unbalance. Haplotype TGGT plays a protective role in Chinese population, while CAGT is a dangerous haplotype of congenital heart disease.
Conclusion: GDF1 gene polymorphism may be related to the susceptibility of Chinese Han congenital heart disease (CHD), especially some subtypes of CTDs. The third part of Chinese congenital heart disease Nodal, GDF1 gene interaction study
Objective: congenital heart disease is a complex genetic disease, which involves complex interactions between genes and genes.Nodal and GDF1 and NODAL signaling ligand, which can interact and affect the regulation of target gene expression by NODAL signals. This study explored the two genetic polymorphisms in Chinese Han congenital The interaction and the pattern of action in sexual heart disease.
Methods: Multifactor Dimensionality Reduction (MDR) was used to analyze the interaction between Nodal and GDF1 genes, and the interaction relationship between genes and genes of SNP and the best interaction model were confirmed by using the interaction tree and interaction map of the interaction system.
Results: the interaction analysis between four polymorphic loci of GDF1 showed that the model composed of 4 polymorphic loci was the best model for interaction, not only P0.0001, the ten cross validation CVC was the most consistent (10/10), and the test accuracy was the maximum (61%); the analysis of the interaction between Nodal and GDF1 was found by GDF1rs7250622, R The 4 factor of s4808870, rs2075762 and Nodal rs2279253 is the best interaction model. The CVC value is 8/10 and the test accuracy is 57.23%. The 3 factor composed of GDF1rs7250622, rs4808867 and rs2075762 is the best interaction model in the conical arterial stem deformity group, and the interaction map shows that Nodal rs2279253 and rs4808870 have significant synergistic effects. The effect of IG was 0.98%, while in the non conical stem deformity group, the 4 factor composed of GDF1rs7250622, rs4808870, rs2075762 and Nodal rs2279254 was the best interaction model. The interaction map analysis showed that there was no interaction between Nodal rs2279253 and rs2279254 and most of the other sites.
Conclusion: there is a significant synergistic effect between the four polymorphic loci of GDF1, and there is a certain synergistic effect between the GDF1 gene and the Nodal gene in the congenital heart disease; the synergistic effect between Nodal rs2279253 and GDF1rs4808870 may have a certain effect on the occurrence of conical arterial stem deformity; the two polymorphic loci of Nodal are in the non conical arterial stem deformity. The disease plays a separate role and does not play a leading role in the interaction effect.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R725.4

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