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

葡萄胎(HM)是一种异常发育的妊娠，滋养细胞过度增殖， 没有胎儿。比较常见的散发性完全性葡萄胎是46，XX或46，XY 雄激素发育概念，其中所有的遗传物质都是父系派生的(AnCHM)。罕见的复发 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中印记基因表达异常 印迹控制区(ICR)上CpG岛的甲基化。最近，常染色体隐性突变在 NLRP7编码一种蛋白(NLRP7)，该蛋白在先天免疫和细胞凋亡中发挥作用。 患有反复BiHM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患者的基因突变可能是印迹形成的主要调节因素。然而，它目前并不是 知道NLRP7是否以及如何执行这一额外功能。此外，它还没有被 研究了NLRP7是否直接与DNA或染色质复合体中的蛋白质相互作用 印记标志的建立或维护。因此，该项目的首要假设是 NLRP7调节在人类活动期间设置的印记标记的重新编程和/或维护 在ICR上通过与DNA和/或染色质修饰因子直接相互作用的卵子发生。因为NLRP7没有 啮齿动物的同源基因，不能在小鼠中产生失活突变。因此，我们建议在体外使用 化验和细胞培养系统在三个具体目标上探索这一假说。在具体目标1中，我们将 通过两种互补的方法进行研究，即电迁移率移位分析和染色质免疫沉淀， NLRP7是否与ICR上甲基化和非甲基化的CpG序列相关联。为实现特定目标2，我们 将与NLRP7进行酵母双杂交相互作用研究和共亲和纯化实验 参与重新编程和印迹维持的候选蛋白质。为了实现具体目标3，我们将 用饱和酵母双杂交筛选全长NLRP7蛋白寻找新的NLRP7相互作用元件 和NLRP7-亮氨酸富含重复区域。对于所有这三个具体目标，我们首先将重点分析已知 或在印记中起作用的新的相互作用因素。然而，如果数据不支持印记中的直接作用， 这些实验将能够解决另一个假设，即在 BiHM次要于破坏NLRP7在生殖系统免疫反应中的更一般作用 器官和/或发育中的卵母细胞通过关注这些途径的候选蛋白质。 总体而言，我们的目标是探索新的途径，以NLRP7为中心，这些途径对印记和 生殖健康。因为一些患有复发性BiHM的妇女罕见的非磨牙妊娠会受到 流产、宫内发育迟缓或早产，我们预测这个项目将揭示 这些常见生殖疾病的候选基因，以及潜在的新治疗靶点。我们将研究一种名为NLRP7的基因的新功能，该基因在罕见复发的女性中发现发生突变 葡萄胎(一种严重的妊娠并发症，伴有胎盘增生和胎儿缺如)，以及 有时还会出现其他妊娠并发症。因为这些包虫的遗传印记是不正常的 Moles，我们建议确定该基因是否在印迹调节中发挥作用。这个项目有 可能导致对遗传印记障碍和产科病因的新理解 并发症和一般的妊娠丢失。

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.