The role of NLRP7 and related genes in hydatidiform moles and reproductive failur

NLRP7及相关基因在葡萄胎和生殖障碍中的作用

• 批准号: 7882072
• 负责人: IGNATIA B VAN DEN VEYVER
• 金额: $ 0.75万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882072
• 关键词: 19q13.42; Address; Affect; Affinity Chromatography; Apoptosis; Biological Assay; Candidate Disease Gene; Cell Culture System; Chromatin; Chromosomes; Chromosomes, Human, Pair 13; Complete Hydatidiform Moles; Complex; Conceptions; CpG Islands; Cultured Cells; DNA; DNA Methylation; Data; Defect; Discipline of obstetrics; Disease; Family; Female; Fetus; Funding Mechanisms; Gene Silencing; Genes; Genetic Materials; Genome; Genomic Imprinting; Goals; Growth; Human; Hydatidiform Mole; Hyperplasia; Immune; Immune response; Inherited; Karyotype; Length; Leucine-Rich Repeat; Maintenance; Methods; Methylation; Mus; Mutate; Mutation; Natural Immunity; Oocytes; Oogenesis; Organ; Pathology; Pathway interactions; Pattern; Placenta; Play; Pregnancy; Pregnancy Complications; Pregnancy loss; Premature Birth; Proteins; Recurrence; Regulation; Reproductive Health; Research; Risk; Rodent; Role; Secondary to; Spontaneous abortion; Tissues; Woman; X Chromosome; Yeasts; base; chromatin immunoprecipitation; demethylation; genome-wide; imprint; in vitro Assay; insight; member; natural Blastocyst Implantation; new therapeutic target; novel; overexpression; protein function; reproductive; research study; trophoblast; yeast two hybrid system

Hydatidiform moles (HM) are abnormally developing pregnancies with hyperproliferative trophoblast and absence of a fetus. The more common sporadic complete hydatidiform moles are 46,XX or 46,XY androgenetic conceptions, in which all the genetic material is paternally derived (AnCHM). Rare recurrent hydatidiform moles are clinically and pathologically identical to AnCHM, but have normal biparental inheritance (BiHM) and their molar trophoblast tissues show abnormal expression of imprinted genes and abnormal methylation of CpG islands at imprinting control regions (ICRs). Recently, autosomal recessive mutations in NLRP7, encoding a protein (NLRP7) with a putative role in innate immunity and apoptosis, were identified in women with recurrent BiHM pregnancies. Prior to this surprising finding, we and others hypothesized that the gene mutated in women with BiHM should be a major regulator of imprinting. However, it is currently not known whether and how NLRP7 might carry out this additional function. Furthermore, it has not yet been studied whether NLRP7 interacts directly with DNA or proteins in chromatin complexes that regulate establishment or maintenance of imprinting marks. Therefore, the overarching hypothesis for this project is that NLRP7 regulates reprogramming and/or maintenance of imprinting marks that are set during human oogenesis by direct interaction with DNA and/or chromatin modifying factors at ICRs. Because NLRP7 has no rodent orthologue, inactivating mutations in mice cannot be generated. Hence, we propose to use in vitro assays and cell culture systems to explore this hypothesis in three specific aims. In specific aim 1 we will investigate by two complementary methods, electromobility shift assays and chromatin immunoprecipitation, whether NLRP7 associates with methylated and unmethylated CpG sequences at ICRs. For specific aim 2 we will perform yeast-two-hybrid interaction studies and co-affinity purification experiments of NLRP7 with candidate proteins that participate in reprogramming and maintenance of imprinting. For Specific aim 3 we will search for novel NLRP7 interactors by a saturated yeast-two-hybrid screen with the full-length NLRP7 protein and the NLRP7-leucine-rich repeat region. For all three specific aims, we will first focus our analysis on known or novel interactors that play a role in imprinting. However, if the data do not support a direct role in imprinting, the experiments, will be able to address an alternate hypothesis, which is that the imprinting defects seen in BiHM are secondary to disruption of a more general role of NLRP7 in immune response within reproductive organs and/or the developing oocyte by focusing on candidate proteins for these pathways. Overall, our goal is to explore new pathways, centered on NLRP7 that are important for imprinting and for reproductive health. Because some women with recurrent BiHM have rare non-molar pregnancies affected by miscarriage, intra-uterine growth retardation or preterm delivery, we predict that this project will uncover candidate genes for these common reproductive disorders, and potentially novel therapeutic targets. We will investigate a new function of a gene, NLRP7, found to be mutated in women who have rare recurrent hydatidiform moles (a severe pregnancy complication with hyperplastic placenta and absent fetus), and sometimes other pregnancy complications. Because genetic imprinting is abnormal in these hydatidiform moles, we propose to determine whether this gene play a role in the regulation of imprinting. This project has the potential to result in new understanding of genetic imprinting disorders and causes of obstetrics complications and pregnancy loss in general.

葡萄胎 (HM) 是滋养细胞过度增殖和发育异常的妊娠。 没有胎儿。更常见的散发性完全葡萄胎是 46,XX 或 46,XY 雄激素受孕，其中所有遗传物质均源自父系 (AnCHM)。罕见复发 葡萄胎在临床和病理上与 AnCHM 相同，但具有正常的双亲遗传 （BiHM）及其磨牙滋养层组织显示印迹基因的异常表达和异常 印记控制区 (ICR) 的 CpG 岛甲基化。最近，常染色体隐性突变 NLRP7 编码一种在先天免疫和细胞凋亡中具有推定作用的蛋白质 (NLRP7)，在 BiHM 反复妊娠的妇女。在这一令人惊讶的发现之前，我们和其他人假设 BiHM 女性中突变的基因应该是印记的主要调节因子。不过，目前还没有 已知 NLRP7 是否以及如何执行此附加功能。此外，目前还没有 研究了 NLRP7 是否直接与调节染色质复合物中的 DNA 或蛋白质相互作用 压印标记的建立或维护。因此，该项目的总体假设是 NLRP7 规范对人类过程中设置的印记标记的重新编程和/或维护 通过与 ICR 处的 DNA 和/或染色质修饰因子直接相互作用来产生卵子。因为 NLRP7 没有 啮齿类动物的直系同源物，不能在小鼠中产生失活突变。因此，我们建议使用体外 分析和细胞培养系统在三个特定目标中探索这一假设。在具体目标 1 中，我们将 通过两种互补方法进行研究，即电动迁移率测定和染色质免疫沉淀， NLRP7 是否与 ICR 处的甲基化和非甲基化 CpG 序列相关。对于具体目标 2，我们 将进行酵母-两种杂交体相互作用研究和 NLRP7 的共亲和纯化实验 参与重编程和印记维持的候选蛋白质。对于具体目标 3，我们将 通过使用全长 NLRP7 蛋白的饱和酵母双杂交筛选寻找新型 NLRP7 相互作用因子 以及 NLRP7 富含亮氨酸的重复区域。对于所有三个具体目标，我们将首先将分析重点放在已知的 或在印记中发挥作用的新颖互动者。然而，如果数据不支持印记的直接作用， 这些实验将能够解决另一个假设，即在 BiHM 是继发性破坏 NLRP7 在生殖系统免疫反应中的更普遍作用的结果。 通过关注这些途径的候选蛋白质来研究器官和/或发育中的卵母细胞。 总的来说，我们的目标是探索以 NLRP7 为中心的新途径，这对于印记和 生殖健康。因为一些患有复发性 BiHM 的女性很少有非葡萄胎妊娠， 流产、宫内发育迟缓或早产，我们预测该项目将揭示 这些常见生殖疾病的候选基因，以及潜在的新治疗靶点。我们将研究 NLRP7 基因的新功能，该基因在患有罕见复发性乳腺癌的女性中发现突变。 葡萄胎（一种严重的妊娠并发症，伴有胎盘增生和胎儿缺失），以及 有时还有其他妊娠并发症。因为这些葡萄胎的基因印记是异常的 鼹鼠，我们建议确定该基因是否在印记调节中发挥作用。该项目有 有可能对遗传印记疾病和产科病因产生新的认识 一般并发症和流产。