Functional characterization of regulatory variants associated with maternal hyperglycemia

项目摘要 孕妇高血糖和妊娠期糖尿病(GDM)每年导致17%的妊娠并发症 导致母亲和孩子的严重急性和终生围产期结局。一个全基因组的 对4,437名孕妇进行的关联研究建立了与妊娠期血糖相关的基因座 特征。重要的关联包括那些特定于怀孕的关联以及之前涉及的几个关联 在非妊娠人群中具有代谢特征。这项建议的目的是量化 数千名母亲高血糖相关基因变异对基因调控的影响。我们满足以下要求的方法 这一目标将使用种群规模的高通量报告分析来确定所有变体的效果 在五个母亲高血糖中，GWA基因座对基因调节的作用以及针对性的基因组编辑策略 和额外的基因组分析。我们已经完成了对这些区域的定向捕获和测序 针对来自四个祖先的835名母亲，她们在种族的极端年代表现出新陈代谢表型 一群人。 我的假设是，这些区域中常见的和低频的遗传变异都有助于 母亲高血糖和因果变异的功能后果将是基因改变 表情。我将从三个具体目标来检验这一假设。在目标1中，我将使用高吞吐量的记者 我们实验室开发了一种方法来量化假定的增强子和因果变异在母体中的影响 高血糖基因座对基因调控的影响。直接检测供体DNA的优点是能够识别 不仅是常见的，而且是罕见的调控变体，并与表型直接相关。在《目标2》中，我将绘制地图 增强子的远程物理关联以确认其目标基因。结果将是第一个 人类基因组基因座三维顺式调控结构的完整解剖 代谢紊乱。在目标3中，我将使用基于CRISPR/Cas9的基因组和表观基因组编辑工具来 从功能上验证预期通过改变增强子活性来扰乱基因表达的因果变异。 预期的结果是，我将确定导致母体高血糖的顺式调控框架 并在患者队列中展示了一种新的可移植的GWAS筛查方法。这将会有 对识别有代谢性疾病风险的母亲和未来新途径的直接积极影响 学习。

Project Summary Maternal hyperglycemia and gestational diabetes mellitus (GDM) complicate 17% of pregnancies annually resulting in serious acute and lifelong perinatal outcomes for both mother and child. A genome-wide association study (GWAS) of 4,437 pregnant mothers established loci associated with gestational glycemic traits. Significant associations included those specific to pregnancy as well as several previously implicated with metabolic traits in non-gravid populations. The objective of this proposal is to quantify the effect of thousands of maternal hyperglycemia associated genetic variants on gene regulation. Our approach to meet this objective will use a population-scale high-throughput reporter assay to determine the effect of all variants in five maternal hyperglycemia GWA loci on gene regulation followed by targeted genome editing strategies and additional genomic assays. We have already completed targeted capture and sequencing of these regions for 835 mothers across four ancestries who exhibited metabolic phenotypes in the extreme deciles of their race group. My hypothesis is that both common and low frequency genetic variants in these regions contribute to maternal hyperglycemia and that the functional consequence of the causal variants will be altered gene expression. I will test that hypothesis in three specific aims. In Aim 1, I will use a high-throughput reporter approach developed by our lab to quantify the impact of putative enhancers and causal variants in maternal hyperglycemia loci on gene regulation. The advantage of assaying donor DNA directly is the ability to identify not only common, but also rare regulatory variants and relate them directly with phenotype. In Aim 2, I will map the long-range physical associations of enhancers to confirm their target genes. The outcome will be the first complete dissection of the three-dimensional cis-regulatory architecture of genomic loci implicated in a human metabolic disorder. In Aim 3, I will use CRISPR/Cas9-based genome and epigenome editing tools to functionally validate causal variants expected to perturb gene expression through altering enhancer activity. The expected outcomes are that I will identify the cis-regulatory framework driving maternal hyperglycemia and demonstrate a novel transferable GWAS screening methodology in a patient cohort. This will have immediate positive impacts on identifying mothers at risk for metabolic disease and new pathways for future studies.