Identifying Molecular Signatures of Genomic Imprinting Errors

识别基因组印记错误的分子特征

基本信息

批准号：
10595043
负责人：
Mellissa Rae Wigle Mann
金额：
$ 37.42万
依托单位：
MAGEE-WOMEN'S RES INST AND FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10595043
关键词：
Affect Assisted Reproductive Technology Biological Biological Markers Biopsy Cells Chemicals Child Clinic Couples DNA Methylation Data Defect Development Disease Embryo Embryonic Development Embryonic Induction Epigenetic Process Fetal Growth Retardation Frequencies General Population Genes Genetic Genomic Imprinting Germ Cells Human Impairment Importins Incidence Incubated Infertility Knowledge Lead Low Birth Weight Infant Maintenance Mammals Mediating Medical Methods Methylation Modality Molecular Profiling Mus Mutagenesis North America Nuclear Import Oocytes Oogenesis Outcome Pathway interactions Population Prader-Willi Syndrome Pre-implantation Embryo Development Predisposition Premature Birth Procedures Proteins Proxy Publishing Regulation Reporter Reporting Research Proposals Risk Screening procedure Silver Silver-Russell syndrome Superovulation TRIM Gene Testing Transcript Unsafe Sex bisulfite blastocyst blastomere structure clinical practice embryo cell embryo culture imprint improved infertility treatment nucleocytoplasmic transport pharmacologic predictive marker preimplantation protein degradation single-cell RNA sequencing transcriptome sequencing trophoblast trying to conceive whole genome

项目摘要

Abstract Alarmingly, ~50 million couples worldwide are unable to conceive after 5 years of unprotected sex. Medically assisted reproductive technologies (ARTs) constitute important treatment modalities for infertile couples trying to conceive. While generally considered safe, ARTs are associated with higher incidences of preterm birth, intrauterine growth restriction, and low birth weight. Moreover, there are more Beckwith-Wiedemann, Silver- Russell, Angelman and Prader-Willi Syndrome children in the ART population harboring imprinted methylation errors compared to those in the general population. Since ART usage coincides with important stages of imprinted DNA methylation programming during gamete and preimplantation development, it is possible that ARTs lead to imprinting errors. Studies in fertile mice support this. Superovulation during oogenesis and/or embryo culture during preimplantation, two nearly universal ART procedures, lead to significant numbers of embryos with imprinted methylation errors. To date, little is known about the regulation of imprinted methylation maintenance during preimplantation development, and moreover, how ARTs lead to disruption in this maintenance. This proposal will address these knowledge gaps. In Aim 1, we will test the hypothesize that embryos are predisposed to imprinted methylation errors because ARTs disrupt crucial maternal-effect transcripts in oocytes that are required in embryos to maintain imprinted methylation. Candidate maternal- effect transcripts will be identified using single cell RNA-seq on control and superovulated oocyte, 1-cell and 2- cell embryos, representing the period of oocyte-to-embryo transition. Candidate function will then be determined though protein degradation methods. Next, we will determine whether altered maternal-effect transcripts in polar bodies, as a proxy for oocytes, correlates with imprinted methylation errors in resulting embryos, ultimately leading to identification of transcript biomarkers that are predictive of imprinted methylation errors. Aim 2 will test the importance of nuclear import in imprint maintenance using pharmacological and protein degradation methods, and whether it is disrupted by ARTs. In Aim 3, we hypothesize that the 4 to 8-cell stages represent a window of susceptibility when fast developing ART embryos acquire DNA methylation errors, particularly in outer, trophoblast cells. We will determine when embryo development is altered by ARTs using time-lapse analysis, and then compare DNA methylation perturbation in slow and fast developing embryos using whole genome bisulfite mutagenesis. This will distill a molecular signature of DNA methylation errors. Next, we will investigate this molecular signature during early development to determine when DNA methylation errors arise and in which lineages of ART embryos, as well as in cells induced to trophoblast fate. Finally, we will test the predictive power of developmental and morphokinetic parameters as a noninvasive procedure for embryos with a molecular signature of imprinted methylation errors. Results from this proposal will provide the biological basis for improvements to fertility treatments aimed at identifying at-risk embryos.

抽象的令人震惊的是，全世界约有5000万对夫妇经过5年的无保护性行为无法构想。医学上辅助生殖技术（艺术）构成了不育夫妇的重要治疗方式受孕。虽然通常被认为是安全的，但艺术与早产的更高发生率有关宫内生长限制和低出生体重。此外，还有更多的贝克维斯·威德曼，银 - 罗素（Russell），安吉尔曼（Angelman）和普拉德·威利（Prader-Willi）综合症儿童在艺术人群中，藏有印迹的甲基化与普通人群中的错误相比。由于艺术用法与重要阶段相吻合配子和植入前开发过程中的印迹DNA甲基化编程，有可能艺术导致烙印错误。肥沃小鼠的研究支持这一点。卵子发生和/或期间的超排量植入前的胚胎培养，两种几乎普遍的艺术程序，导致大量具有印记甲基化误差的胚胎。迄今为止，关于印迹甲基化的调节知之甚少在植入前开发期间的维护，此外，艺术如何导致这一中断维护。该建议将解决这些知识差距。在AIM 1中，我们将测试假设的胚胎倾向于印迹甲基化误差，因为艺术破坏了至关重要的母体效应卵母细胞中所需的卵母细胞的转录本维持印迹甲基化。候选母亲将使用单细胞RNA-seq在对照和超卵形卵母细胞，1细胞和2-- 细胞胚胎，代表卵母细胞到胚胎过渡的时期。候选功能将是确定蛋白质降解方法。接下来，我们将确定是否改变了产妇效应极性物体中的成绩单，作为卵母细胞的替代品，与产生的甲基化误差相关胚胎，最终导致鉴定转录生物标志物，这些生物标志物可预测印迹甲基化错误。 AIM 2将测试使用药理和蛋白质降解方法，以及它是否受到艺术的破坏。在AIM 3中，我们假设4到8个细胞快速开发Art Embryos获取DNA甲基化时，阶段代表了易感性的窗口误差，特别是在外部滋养细胞中。我们将确定何时改变胚胎的发展使用延时分析，然后比较缓慢而快速发展的DNA甲基化扰动使用全基因组硫酸诱变的胚胎。这将提炼DNA甲基化的分子特征错误。接下来，我们将在早期发育过程中研究该分子特征，以确定何时DNA 出现甲基化误差，其中艺术胚胎的谱系以及诱导滋养细胞命运的细胞中。最后，我们将测试发育和形态运动参数的预测能力作为无创的具有印记甲基化误差的分子特征的胚胎的程序。该提议的结果将为旨在识别高风险胚胎的生育治疗改善生物学基础。