Functional characterization of regulatory variants associated with maternal hyperglycemia

项目摘要 产妇高血糖和妊娠糖尿病（GDM）每年使17%的妊娠并发症 导致母亲和婴儿严重的急性和终身围产期后果。全基因组 一项对4,437名孕妇的关联研究（GWAS）确定了与妊娠期血糖相关的基因座， 性状重要的关联包括那些特定于怀孕以及一些以前涉及的 在非妊娠人群中具有代谢特征。本提案的目的是量化 成千上万的母体高血糖相关的基因变异对基因调控的影响。我们的会面方式 该目标将使用群体规模的高通量报告分析来确定所有变体的影响 在五个母体高血糖GWA基因座上进行基因调控，然后进行靶向基因组编辑策略 和其他基因组测定。我们已经完成了对这些区域的靶向捕获和测序 研究对象是来自四个祖先的835位母亲，她们在种族的极端十分位数中表现出代谢表型， 组 我的假设是，这些区域的常见和低频率遗传变异都有助于 母亲的高血糖症和功能后果的因果变异将改变基因 表情我将在三个具体目标中检验这一假设。在目标1中，我将使用高通量报告器 我们的实验室开发的方法，以量化推定的增强子和因果变异的影响，在母体 高血糖位点对基因调控的影响。直接分析供体DNA的优点是能够识别 不仅是常见的，而且是罕见的调节变体，并将它们与表型直接相关。在目标2中，我将绘制 增强子的长距离物理关联以确认其靶基因。结果将是第一个 完整解剖涉及人类的基因组位点的三维顺式调控结构， 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， 在功能上验证预期通过改变增强子活性来干扰基因表达的致病变体。 预期的结果是，我将确定顺式调节框架驱动产妇高血糖症 并在患者队列中展示了一种新的可转移GWAS筛选方法。这将具有 对识别有代谢疾病风险的母亲的直接积极影响和未来的新途径 问题研究

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.