Molecular Causes of Down Syndrome Associated Congenital Heart Disease and Other Phenotypes

摘要 唐氏综合征(DS)或21三体与多种发育异常相关，影响 心血管、血液、呼吸系统、免疫学、内分泌、胃肠和神经系统。 额外的21号染色体(CHR)拷贝产生这些系统性问题的机制仍然存在 人们对此知之甚少。我们之前招募了600名先天性心脏病(CHD)DS患者和100名DS患者 无冠心病的DS患者。通过对手术修复过程中丢弃的CHD组织进行分析，我们发现 非整倍体和双倍体冠心病患者心脏基因表达的差异。包括在内 其中，Sost(在Chr 17上编码硬化素)在DS中的表达是在DS中的10倍。 整倍体CHD组织。由于硬化素抑制WNT和BMP信号，我们假设失调可能 在一些DS患者中可能导致CHD。硬化素也影响骨密度，并可能有助于骨骼和 DS的其他异常。我们建议扩大这些分析以确定异常基因的遗传基础。 影响DS表型的表达。 我们假设，对21三体的分子反应受到额外的基因类型的影响 导致患者特有的DS表型。我们将定义这些基因类型及其相关的调控网络 改变细胞和器官发育的物质。我们将利用~700DS的全基因组序列(WGS) 具有不同DS表型的患者将可用于拟议的研究。我们正在生成诱导式 200例DS患者的多能干细胞(IPSCs)。WGS和IPSC派生得到其他资金的支持 机械装置。 我们建议分析WGS以定义与DS表型相关的序列元件。我们会 利用DS、iPSCs和细胞谱系探索这些关联的转录和表观遗传学后果 区别于iPSCs。通过将DS iPS细胞分化为心肌细胞(IPSC-CMS)， 内皮细胞、神经细胞和其他细胞将定义序列对基因表达的影响。我们会 还利用增强的CRISPR/Cas9工程和IPSCs的单细胞分析来研究该基因 与DS表型相关的调控机制。我们还将探讨DS的后果是否- 与整倍体细胞相关的变异，以确定三体是否需要条件性基因型。我们建议 验证DS患者IPSCs和废弃的整倍体和三体21心脏组织的发现。 为了实现这些广泛的目标，我们将：1)使用全基因识别与DS表型相关的遗传变异 基因组序列和RNA表达的eQTL。2)比较单细胞/核转录和ATACseq DS和整倍体iPSCs、分化细胞和CHD组织的图谱。3)定义基因和调控途径 通过干扰DS iPSCs和细胞系中的基因表达来调节DS的表型。

ABSTRACT Down syndrome (DS) or trisomy 21 is associated with multiple developmental anomalies affecting the cardiovascular, hematological, respiratory, immunological, endocrine, gastrointestinal and neurological systems. The mechanisms by which an additional copy of chromosome (Chr) 21 produces these systemic issues remains poorly understood. We have previously recruited 600 DS patients with congenital heart defects (CHD) and 100 DS patients without CHD. Using analyses of discarded CHD tissues obtained during surgical repair, we identified unexpected and distinct cardiac gene expression differences between euploid and DS CHD patients. Included among these we found that SOST (encoding sclerostin on Chr 17) expression was 10-fold higher in DS than in euploid CHD tissues. As sclerostin inhibits WNT and BMP signaling, we hypothesis that dysregulation may contribute to CHD in some DS patients. Sclerostin also impact bone density and may contribute to skeletal and other abnormalities in DS. We propose to expand these analyses to define genetic basis for abnormal gene expression that influence DS phenotype. We hypothesize that the molecular response to trisomy 21 are influenced by additional genotypes that result in patient-specific DS phenotypes. We will define these genotypes and their related regulatory networks that alter cell and organ development. We will capitalize on whole genome sequences (WGS) from ~700 DS patients with different DS phenotypes that will be available for the proposed studies. We are generating induced pluripotent stem cells (iPSCs) from 200 DS patients. WGS and iPSC derivation are supported by other funding mechanisms. We propose to analyze WGS to define sequence elements associated with DS phenotypes. We will explore the transcriptional and epigenetic consequences of these associations using DS iPSCs and cell lineages differentiated from iPSCs. Through the differentiation of DS iPS cells into cardiomyocytes (iPSC-CMs), endothelial, neural cells and others will define the consequences of sequences on gene expression. We will also use enhanced CRISPR/ Cas9 engineering and single cell analyses of iPSCs to investigate the gene regulatory mechanisms associated with DS phenotypes. We will also explore if the consequences of DS- associated variants with in euploid cells, to determine if trisomy is required conditioning genotype. We propose to validate finding using DS patient iPSCs and discarded euploid and trisomy 21 cardiac tissues. To achieve these broad goals, we will: 1) Identify genetic variants associated with DS phenotypes using whole genome sequence and RNA expression eQTLs. 2) Compare single cell/nucleus transcriptional and ATACseq profiles of DS and euploid iPSCs, differentiated cells, and CHD tissues. 3) Define genes and regulatory pathways that modulate DS phenotypes by perturbing gene expression in DS iPSCs and cell lineages.