Ano5突变抑制PS暴露影响骨代谢平衡导致FGC发生的机制研究

家族性巨大型牙骨质瘤（FGC）是一种罕见的常染色体显性遗传病，严重的颌面畸形及口腔功能障碍极大影响患者的身心健康，但至今仍未明确其致病基因。申请人前期临床发现一个迄今报道黄种人中最大FGC家系，对患病者进行全外显子测序，发现致病基因ANO5及突变位点，并在Ano5基因敲除小鼠模型上证实其骨矿化能力下降、破骨细胞形成障碍；进一步RNA-Seq验证Ano5敲除后成骨相关基因表达下调、质膜外侧相关基因改变及代谢通路异常。同时ANO5作为磷脂爬行酶能影响质膜内侧的磷脂酰丝氨酸（PS）暴露于质膜外，由此推测Ano5突变后通过抑制PS暴露影响骨代谢平衡导致FGC的发生。本研究从三个方面：①完善Ano5突变的骨代谢失衡模型；②明确Ano5突变后通过抑制PS暴露影响骨沉积及破骨细胞形成；③筛选Ano5敲除小鼠致病的关键节点和作用因子；拟进一步阐明ANO5在骨代谢中的分子机制，为临床治疗FGC提供候选靶点。

Familial Gigantiform Cementoma (FGC) is a rare autosomal dominant ossifying fibroma. Progressively expansive lesions of the jaws can be massive and cause remarkable facial deformity, thus it threatens the physical and mental health of patients. To date, the exact cytogenetic and molecular genetics of this disease remained unknown. In preliminary studies, we have first reported a large Chinese FGC family with thirteen affected members suffered from Gigantiform Cementoma. Further Whole Exome Sequencing (WES) revealed the heterozygous mutation in ANO5 gene (c.1538C > T, p.Thr 513 Ile) in these patients. We next used Ano5-deficient mice, which appeared to impaired osteoblastogenesis and osteoclastogenesis. While Ano5 KO osteoblast cultures showed decreased mineral deposition and osteoclasts resulted in disrupted cell fusion. By carrying out RNA sequencing (RNA-seq) between Ano5+/+ and Ano5-/- mice, we have systematically screened for novel molecules and observed lower expression of osteoblast-related genes, external side of plasma membrane genes as well as genes with the greatest fold change of metabolic pathways. ANO5 may function as a phospholipid scramblase at inner membranes and to facilitate translocation of phospholipid between membrane leaflets. Based on these novel observations, we hypothesize that the mutation in Ano5 disrupts phospholipid exposure on bone metabolism resulting in FGC. To address this we aim to: ① Continue to characterize the phenotype of impaired bone metabolism conditional Ano5 mutant mice; ② Validate Ano5 mutation causing a lose-of-function effect associated with regulatory dysfunction of PS exposure contributing to the regulation of bone mass in vitro; ③ Screen out genome regulatory networks and potential critical node on diseased Ano5 KO mice. This project will focus on the relevance of molecular mechanism of Ano5 in bone metabolism, and may provide additional choice for targeting therapy of FGC.