【作者】 包碧惠；

【导师】 梁志清；

【作者基本信息】 第三军医大学 ， 妇产科学， 2013， 博士

【摘要】 研究背景先天性心脏畸形(CHD)是人类最常见的出生缺陷，也是胎婴儿死亡的主要原因。而大多数CHD的病因目前尚不清楚，仅少数由单基因突变和染色体畸变引起，多基因遗传和环境因素相互作用是心血管发育缺陷的主要病因。研究CHD的遗传因素和表观遗传调控机制，进一步从本质上认识和诊断疾病，促进CHD的早期产前筛查和有效预防，提高出生人口素质。基因拷贝数变异(CNV)是一种常见的亚显微的遗传改变，染色体微缺失或微重复改变基因剂量使功能效应受损。参与心脏发育的基因在心肌细胞的增殖、分化和功能的发挥中起重要的作用，这些位点的亚显微缺陷将引起心脏发育异常。全基因拷贝数分析可识别一系列靶向基因座的亚显微缺失和重复，微阵列为基础的方法学为先天性心脏畸形的遗传变异提供一个广泛的基因组调查，并可靠地识别致病性的基因组序列不平衡，表型相关的基因信息也将进一步得到阐释。DNA甲基化修饰作为表观遗传调控的重要组成部分，对胚胎的正常发育具有显著作用，其修饰异常将会引起心血管的发育失调,甚至胚胎死亡。人们日常生活暴露水平的一些危害健康的环境因素、营养因素可增加基因的不稳定性和改变组织细胞的物质代谢，改变DNA甲基化模式的动态平衡将对胚胎和胎儿发育产生严重的后果。表观遗传学揭示环境因素如何影响基因的选择性表达，对传统遗传学难以解释的CHD进入表观调控的分子研究，将为积极有效预防胎婴儿的心血管缺陷提供新的科学依据。目的分析心脏畸形胎儿基因拷贝数变异和甲基化修饰情况，研究基因拷贝数变异体、基因启动子区甲基化差异在心脏畸形可能的致病机制，为寻找新的生物分子标记促进心血管畸形的早期产前诊断提供实验依据。方法1.应用Affymetrix SNP6.0芯片对10个样本进行全基因拷贝数分析，包括核型正常的先心胎儿5例、染色体畸变先心胎儿2例及正常胎儿1例，以HapMap90个亚洲人群为参照，筛查、分析DNA拷贝数异常的区域，挑选最有生物信息的染色体区域；2.筛选与胚胎心脏发育相关的基因以及可能涉及心脏形成的候选基因的拷贝数变异体，采用罗氏LightCycler自动定量PCR分析仪进行验证，与父母、正常新生儿及健康志愿者对照。3.运用Roche-NimbleGen人HG18Meth3×720K甲基化芯片对复杂先心胎儿进行全基因组DNA甲基化水平分析，将心内膜垫缺损、单房室瓣和大动脉转位的胎儿3例与孕周、性别匹配的正常胎儿3例进行对比，NimbleScan软件分析并图形化两组样本之间的DNA甲基化水平的差异及差异位点的序列信息。4.对有差异的DNA甲基化序列，将相同的实验样本和对照样本进行独立的分析验证，运用Sequenom质谱检测平台进行甲基化定量检测，比较两组样本是否在相同区域存在DNA甲基化差异，初步分析表观遗传调控对胎儿心血管发育的影响。结果1.7例CHD患儿共10份DNA样本进行了全基因拷贝数分析，出现了众多的CNVs，仅部分CNVs包含生物信息，一些可能对心脏发育有重要作用的基因出现了拷贝数重复或缺失，双胎中的患TOF胎儿与正常胎儿拷贝数经后续验证后无显著差异。2. qPCR验证了所有可能与心脏发育相关的CNVs，除了18-三体患儿的GATA6拷贝数重复可能扰乱胚胎心脏发育，14q23.1单拷贝数缺失经反复对比分析得到验证，该区域包含了整个DAAM1和KIAA0666基因，发生在1例核型正常的复杂CHD胎儿，而DAAM1对动物心脏形态发生必不可少，DAAM1拷贝数缺失可能是复杂先心患儿的潜在病因。3.分析复杂心脏畸形胎儿3例和对照组正常胎儿3例的全基因组DNA甲基化水平，发现RADIL、CHRNA10、RADIL基因启动子区甲基化水平高于正常对照组0.41~0.59，而NXPH3、ZNF408和ABL1基因甲基化程度较正常对照组降低0.34~0.62。4.对基因组分布的DNA甲基化差异位点，运用SequenomMassARRAY系统，采用相同实验组和对照组的脐血DNA进行定量甲基化分析验证，未发现以上几个基因对应区域的CpG岛有甲基化水平差异。结论1.发生频率远高于染色体结构变异的基因拷贝数变异促进了人类遗传的多样性，也增加了出生缺陷的风险。2.18-三体频发CHD与Hsa18基因尤其是GATA6过量表达、扰乱了胚胎心血管的分化发育相关。3.遗传位点14q23.1拷贝数缺失，所含的DAAM1基因对胚胎的心脏发育不可或缺，其单拷贝数缺失可导致复杂CHD。4.复杂CHD患儿全基因组范围内的DNA甲基化分析发现几个基因的DNA甲基化水平变化，但后续的质谱定量分析未能验证这些位点的甲基化差异，DNA甲基化修饰的表遗传机制对胎儿CHD调控机制或需加大临床样本量继续探讨。更多还原

【Abstract】 BackgroundCongenital heart defect, referred to as CHD for short, is the most common birthdefects and the main cause of fetal death in human. Causes of most CHDs have not yet beenknown. Only a few CHDs were caused by gene mutations and chromosome aberrations.The major causes of CHD resulted from interaction of multiple genes and environmentalfactors. To research associating genetic factors and epigenetics with CHD will help usfurther undestand CHD and early diagnose it, which could make more efficient screeningand treatment of fetal CHDs for the quality of the newborn.Copy number variation is common in submicroscopic genetic alterations.Chromosome submicroscopic deletions or amplifications will alter gene expression andimpair fetal development. Genes regulating cardiogenesis play an important role inproliferation, differentiation and functions of myocardial cells. Submicroscopicchromosomal imbalances involving cardiac-specific transcription factors will cause severeheart defects. Genome-wide copy number analysis can identify novel submicroscopicdeletions and amplifications associated with heart development. Researchers areincreasingly employing the microarray-based technique for genome-wide screening of copynumber variations which may help identify pathogenic DNA imbalance. Then geneticinformation will be more understanded.DNA methylation is an important modification of epigenetic regulation, which hassignificant functions in embryonic development. Abnormal DNA methylation may causediseases such as cardiovascular malformations and even fetal death. Daily exposure to toxicenvironmental contaminants or trophic factors can trigger genetic changes and influencecellur metabolism, which may disturb dynamic balance of DNA methylation patterns withserious consequences to fetus. To clarify the molecular mechanism of CHDs withtraditional knowledge is not easy, revealing epigenetic regulation of gene expression underlying CHDs may uncover environmental factors effects without changing DNAsequence. That will provide new scientific basis for effective prevention of fetalcardiovascular defects.ObjectiveThe aim of this study is to explore the molecular mechanism of CHDs and to find newmolecular biomarker of fetal cardiovascular malformations by analyses of genomic copynumber variation and DNA methylation changes associated with fetal CHDs. Next we mayprovide experimental evidence for early prenatal diagnosis of fetal CHDs.Methods1. Affymetrix SNP6.0array was used to analyse10samples for detection of genomiccopy number variations, including5CHD fetuses with normal karyotypes,2CHD fetuseswith abnormal chromosome and1normal fetus, compared with HapMap90Asianpopulation. Copy number variants with biological information were selected for furtheranalysis and verification.2. Fluorescence quantitative polymerase chain reaction (qPCR) assay was used toverified copy number deletions and amplifications in CHD fetal DNA, compared with itsparents, normal neonates and healthy volunteers, which contain genes associated withembryonic heart development and possibly related to cardiogenesis.3. Rhoche NimbleGene3×720K CpG Island Plus RefSeq Promoter array was used toanalyses Genome-wide DNA methylation between complex CHD fetuses and normalfetuses. We chose3cases with endocardial cushion defect, single atrioventricular valve andtransposition of the great arteries matched with3normal fetuses with same gestational ageand gender. Data of the microarray were processed using NimbleScan software. DNAmethylation changes with the genomic information can be viewed on sequence graph fileswith NimbleScan software between cases and controls.4. The same case samples and control samples were further tested by SequenomMassARRAY EpiTYPER DNA methylation platform. Quantitative methylationanalysis and validation were focus on the corresponding regions of DNA methylationdifferences between the cases and controls. Effects of epigenetic modifications on fetalcardiovascular malformations were noted in a preliminary analysis.Results1. Seven CHD fetuses total of10samples underwent genome-wide CNV analyses.Many CNVs arised, only some contained biological information. Some gene CNVs wereobserved that have an important role in heart development. The twin fetuses that one is TOFand the other is normal had no difference in copy number by subsequent verification. 2. We verified all CNVs possibly related to heart development by qPCR. In addition tothe18-trisomy with the copy number amplifications of GATA6related to heart defects, onecopy number deletion at14q23.1was validated which contains whole DAAM1andKIAA0666genes. The single-copy deletion existed in one normal karyotype fetus withcomplex CHDs. DAAM1is essential for early embryonic heart development and its copynumber deletion may be a potential cause of complex CHDs for fetuses.3. DNA methylation differences of3cases with complex CHDs when compared to3matched controls were found that Log2-ratio of RADIL, CHRNA10and RADIL genepromoter methylation were higher0.41~0.59than that of the controls, NXPH3, ZNF408and ABL1gene methylation level was lower0.34~0.62than controls.4. The same cases and controls’ umbilical cord blood DNA were further tested bySequenom MassARRAY system for verification of methylation differences. The screeningresults cannot to be validated by the another methylation analysis method.Conclusions1. More frequent copy number variants than chromose abberrations cause humangenetic diversity that also increase the risk of specific birth defects.2. Frequent CHDs in fetus with trisomy18were associated with overexpression ofHsa18genes especially GATA6, which disturbed cardiac differentiation and thedevelopment of the embryonic outflow tract.3. Copy number deletion on the locus of14q23.1will result in complex CHD, wherecontained DAAM1gene that is indispensable for heart development.4. Significant methylation differences were detected in3fetuses with complex CHDby genome-wide screening of methylation changes. But the methylation changes could notbe verified with subsequent mass spectrometry quantitative analysis of methylation withsame samples. Epigenetic mechanisms of DNA methylation in complex CHD may needadvanced study for more clinical samples.更多还原