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

描述(申请人提供)：葡萄胎(HM)是发育异常的妊娠，滋养层细胞过度增殖，无胎儿。更常见的散发性完全性葡萄胎是46，XX或46，XY雄生殖胚胎，其中所有遗传物质都来自父系(AnCHM)。罕见的复发性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)，其葡萄胎滋养层组织中印记基因异常表达和印记控制区(ICR)上CpG岛异常甲基化。最近，在反复妊娠的BiHM患者中发现了NLRP7的常染色体隐性突变，该基因编码一种蛋白(NLRP7)，该蛋白可能在先天性免疫和细胞凋亡中发挥作用。在这一令人惊讶的发现之前，我们和其他人假设，患有BiHM的女性的基因突变应该是印记的主要调节因素。然而，目前还不知道NLRP7是否以及如何执行这一额外功能。此外，目前还没有研究NLRP7是否直接与DNA或染色质复合体中的蛋白质相互作用，这些复合体调节印迹标记的建立或维持。因此，该项目的总体假设是，NLRP7通过与ICRs的DNA和/或染色质修饰因子直接相互作用，调节人类卵子发生过程中设定的印记标记的重新编程和/或维护。因为NLRP7没有啮齿动物的同源基因，所以不能在小鼠身上产生失活突变。因此，我们建议使用体外分析和细胞培养系统来探索这一假设在三个特定的目标。在具体目标1中，我们将通过两种互补的方法，即电迁移率移位分析和染色质免疫沉淀，研究NLRP7是否与ICR上甲基化和非甲基化的CpG序列相关联。针对特定目标2，我们将进行NLRP7与参与重编程和印迹维持的候选蛋白的酵母双杂交相互作用研究和共亲和纯化实验。对于特定的目标3，我们将通过饱和酵母双杂交筛选寻找新的NLRP7相互作用元件，其中包括全长NLRP7蛋白和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在印记中发挥作用的已知或新颖的相互作用因素。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种假设，即在BiHM中看到的印记缺陷是次要的，因为NLRP7在生殖器官和/或发育中的卵母细胞内的免疫反应中更一般的作用是通过关注这些途径的候选蛋白来破坏的。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非常重要。由于一些患有复发性BiHM的妇女有罕见的非磨牙妊娠，受到流产、宫内发育迟缓或早产的影响，我们预测该项目将发现这些常见生殖疾病的候选基因，并可能发现新的治疗靶点。公共卫生相关性：我们将研究一种名为NLRP7的基因的新功能，该基因被发现在患有罕见复发性葡萄胎(一种伴有胎盘增生和胎儿缺失的严重妊娠并发症)的女性中发生突变，有时还会出现其他妊娠并发症。由于基因印迹在这些葡萄胎中是异常的，我们建议确定该基因是否在印迹的调节中起作用。该项目有可能导致对遗传印记障碍以及产科并发症和妊娠丢失的一般原因的新理解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.