本文選題：先天性心臟病 + NODAL信號通路　； 參考：《蘭州大學》2012年博士論文

【摘要】：先天性心臟病(Congenital heart disease,CHD)是最常見的先天缺陷性疾病,也是引起嬰幼兒死亡最常見的原因,其總體發(fā)病率約8‰。目前,大多數(shù)(80%)先心病被認為是由遺傳和環(huán)境等因素共同作用導致的復雜疾病,其中遺傳因素對先心病的發(fā)生起決定性作用。NODAL信號通路是參與心臟不對稱發(fā)育的重要通路之一,有很多證據(jù)表明NODAL通路基因變異與先心病發(fā)病有關。本文就國際先心病研究中所發(fā)現(xiàn)的NODAL通路多態(tài)性問題展開討論,首次探討NODAL通路中重要的兩個基因Nodal和生長/分化因子(Growth/Differentiation factor1, GDF1)多態(tài)性與中國先天性心臟病的相關性問題,并以此進一步分析先心病中的Nodal基因與GDF1基因間的交互作用。 第一部分Nodal基因多態(tài)性與中國漢族先天性心臟病的關聯(lián)研究 目的：Nodal是NODAL信號通路的主要配體,Nodal配體對維持NODAL信號的穩(wěn)定性和作用范圍、保證下游基因的正常表達起著非常重要的作用。研究發(fā)現(xiàn),Nodal基因的兩個單核苷酸多態(tài)性能夠顯著降低Nodal的生物學活性。本研究旨在探討Nodal基因這兩個第二外顯子區(qū)單核苷酸多態(tài)性是否與中國漢族單純性先天性心臟病患者的發(fā)病相關,同時也對與轉錄后基因表達水平調節(jié)有關的3'UTR區(qū)的兩個多態(tài)位點進行了相關性分析。 方法：采用PCR-變性高效液相色譜(PCR-Denaturing High Performance Liquid Chromatography, PCR-DHPLC)和直接測序的方法對308例先心病患者和314例健康對照進行了Nodal基因第二外顯子區(qū)rs1904589和rs10999334及3'UTR區(qū)rs2279253和rs2279254的病例-對照關聯(lián)研究。 結果：Nodal的2個外顯子SNPs位點在中國漢族病例組和對照組中均無多態(tài)性,全部表現(xiàn)為野生基因型GG；Nodal基因3'UTR的兩個多態(tài)性位點rs2279253和rs2279254雖與先心病總體并無相關性,但將先心病分為圓錐動脈干畸形組(conotruncal defects, CTDs)和非圓錐動脈干畸形組(Non-conotruncal defects, Non-CTDs)西大類后分層分析,rs2279253TT基因型與CTDs發(fā)病風險有潛在關聯(lián)性(P=0.029)；隨后在15種先心病亞型中進行的分析表明,rs2279253T和rs2279254G等位基因頻率都與CTDs組中肺動脈閉鎖(pulmonary atresia, PA)顯著相關(P=2.87×10-4,0.002)。肺動脈閉鎖又可以分為伴有室間隔缺損型(pulmonary atresia with ventriucular septal defect, PA+VSD)和室間隔完整型(pulmonary atresia with an intact ventricular septum, PA+IVS)兩種類型。經統(tǒng)計發(fā)現(xiàn),rs2279253位點次等位基因與PA+IVS顯著相關(P=0.001)。按性別分層分析表明兩個位點與先心病無明顯相關性。 結論：我們的研究提示中國漢族群體Nodal基因第二外顯子區(qū)無多態(tài)性,參與中國漢族單純性先心病發(fā)病的可能性很小,但Nodal3'UTR區(qū)域的單核苷酸多態(tài)性,可能與中國漢族人群中CTDs的某些亞型的易感性有關。 第二部分GDF1基因多態(tài)性與中國漢族先天性心臟病的關聯(lián)研究 目的：GDF1也是NODAL信號的配體,可與Nodal通過異源二聚作用形成共配體。作為NODAL信號通路的上游基因,GDF1直接或間接調控通路中下游基因的表達,在心臟左右不對稱發(fā)育中發(fā)揮關鍵作用。本研究旨在探討GDF1基因多態(tài)性是否與中國漢族先天性心臟病(CHD)患者的發(fā)病相關。 方法：利用HapMap和Haploview篩選出GDF1四個tagSNP (rs7250622, rs4808867,rs4808870, rs2075762),采用PCR-DHPLC和PCR-限制性片段長度多態(tài)性(PCR-Restriction Fragment Length Polymorphism, PCR-RFLP)的方法對這四個tagSNP進行先心病病例-對照關聯(lián)研究。 結果：所檢測的GDF1的四個tagSNPs位點在病例組和對照組的基因型頻率分布均符合哈迪-溫伯格平衡。這四個tagSNP在等位基因頻率和基因型的分布上與中國人群總體先天性心臟病的易感性均無相關性；但是,按是否與圓錐動脈干發(fā)育有關對病例進行分層后發(fā)現(xiàn),rs4808870位點AA基因型與CTDs組有關(P=0.027),進一步在先心病亞型中的分析也表明GDF1rs4808870的A等位基因與肺動脈閉鎖相關(P=0.003)；對GDF1基因4個多態(tài)性位點的連鎖不平衡分析中發(fā)現(xiàn),4個位點呈較強連鎖不平衡；單倍型TGGT在中國人群中起保護性作用而CAGT是先心病發(fā)生的危險單體型。 結論：GDF1基因多態(tài)性可能與中國漢族先天性心臟病(CHD),尤其是CTDs某些亞型的易感性有關。第三部分中國先天性心臟病Nodal、GDF1基因的交互作用研究 目的：先天性心臟病屬于復雜性遺傳病,其發(fā)病涉及到基因與基因之間錯綜復雜的相互作用。Nodal和GDF1同屬于NODAL信號通路配體,兩者能夠相互作用,影響NODAL信號對靶基因表達的調控。本研究探討了這兩種基因多態(tài)性在中國漢族先天性心臟病中的交互作用及其作用的模式。 方法：利用多因子降維法(Multifactor Dimensionality Reduction,MDR)分析Nodal和GDF1基因間的交互作用；進一步應用互作系統(tǒng)樹及互作圖譜來分析確證SNP的基因與基因之間的相互作用關系及最佳互作模型。 結果：GDF1四個多態(tài)位點間的互作分析結果顯示,由4個多態(tài)位點共同組成的模型為相互作用的最佳模型,不僅P0.0001、十重交叉驗證CVC的一致性最大(10/10),而且測試精確度最大(61.00%)；對Nodal與GDF1之間的交互作用分析發(fā)現(xiàn),由GDF1rs7250622、rs4808870、rs2075762和Nodal rs2279253組成的4因子為最佳互作的模型,CVC值為8/10,測試精確度為57.23%；在圓錐動脈干畸形組中由GDF1rs7250622、rs4808867、rs2075762組成的3因子為最佳互作的模型,互作圖譜表明Nodal rs2279253與rs4808870有顯著的協(xié)同效應,互作效應IG墑為0.98%；而在非圓錐動脈干畸形組,由GDF1rs7250622、 rs4808870、rs2075762和Nodal rs2279254組成的4因子為最佳互作的模型,互作圖譜分析結果顯示Nodal rs2279253和rs2279254之間及與其他大多數(shù)位點均無互作效應。 結論：GDF1四個多態(tài)位點間有顯著的協(xié)同效應；先心病中GDF1基因與Nodal基因間存在一定的協(xié)同作用；Nodal rs2279253與GDF1rs4808870之間的協(xié)同作用對圓錐動脈干畸形的發(fā)生可能產生一定的影響；Nodal兩個多態(tài)位點在非圓錐動脈干畸形的發(fā)病中單獨起作用,在互作效應中并不占主導地位。
[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.
【學位授予單位】：蘭州大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R725.4

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