ANO5基因突变导致Gnathodiaphyseal dysplasia的发病机制研究

Gnathodiaphyseal dysplasia(GDD)is an autosomal dominantly inherited disorder characterized by cemento-ossifying fibroma, purulent osteomyelitis, cortical thickening of tubular bones and diaphyseal sclerosis. Three missense mutations in the ANO5 gene have been identified in affected members from Japanese, African-American and Italian families respectively. However, GDD-associated muation in Chinese families remains unknown so far. For the first time, we found a novel missense mutation causing the p.Cys360Tyr substitution in a Chinese family with GDD recently. Moreover, evidence showed that ANO5 involved in the function of osteoblasts in vitro. Based on our achievements, we will focus on research into mechanism of the GDD disorder. Firstly, the different effects on protein expression, localization, stability and proliferation caused by mutation which was found in Chinese family will be investigated by site-directed mutagenesis. Secondly, osteoblast differentiation and function will be detected after ANO5 gene is overexpressed in MC3T3 cells. Osteoblast-related factors will be detected and the matrix and mineral formation in osteoblasts will be evaluated by ALP and von Kossa/alizarin red S staining. Thirdly, C360Y knock-in mouse and ANO5 knockout mouse models will be established. All phenotypes related to disorder should be detected. Fourthly, alteration of osteogenesis including matrix and mineral formation and bone-related signaling pathways including TGFβand BMP et al will be studied. Finally, we will investigate if GDD degradation can be rescued by inhibitor of proteasome and the PI3K, and try to clarify the molecular mechanism of GDD.

颌骨和骨干发育不良（Gnathodiaphyseal Dysplasia,GDD)是累及口腔和长骨的常染色体显性遗传病。表现为牙骨质-骨化纤维瘤、颌骨化脓性骨髓炎和长骨骨皮质增厚硬化，严重影响患者生活质量。国外GDD家系中发现3个ANO5基因突变，但尚无中国GDD的报道。申请人近期首次在中国GDD家系中，发现ANO5基因的新错义突变C360Y，体外前期实验提示ANO5与成骨细胞功能有关。本研究拟通过细胞实验和动物模型，探讨ANO5基因突变导致GDD的发病机制。⑴定点诱变观察突变对蛋白定位、稳定性及细胞增殖的影响；⑵在成骨细胞系MC3T3中过表达ANO5基因，观察成骨和矿化功能改变；⑶建立中国C360Y突变敲进和ANO5基因敲除小鼠模型，并检测表型效应；⑷检测缺陷小鼠成骨功能改变，及与成骨相关的TGFβ和BMP等信号通路；⑸利用蛋白酶体和PI3K抑制剂，观察缺陷小鼠ANO5蛋白稳定性的改变。

颌骨和骨干发育不良（GDD) 是一种累及口腔和长骨骨干的常染色体显性遗传病。口腔主要表现为牙骨质-骨化纤维瘤和颌骨骨髓炎；长骨主要表现为骨皮质增厚和骨干硬化，青少年时期可发生自主性骨折，严重影响患者的生活质量。ANO5基因是本病的致病基因。课题组收集到一个中国GDD 家系，并首次发现中国ANO5基因新突变C360Y。为揭示ANO5基因突变导致GDD发生的分子机制，我们从体外和动物模型两个方面进行研究。.体外实验结果显示：1.通过体外定点诱变，发现突变型ANO5蛋白质表达降低，并且与内质网特异性标志物钙网蛋白共表达；2. 利用shRNA将成骨前体细胞MC3T3-E1中的Ano5基因沉默，发现在成骨分化过程中Ano5表达升高，并且成骨和矿化能力明显增高。.利用CRISPR/Cas9技术我们建立Ano5基因敲除（KO）和C360Y突变敲入（KI）小鼠模型。结果发现：1. Ano5 KO小鼠重复出部分GDD患者的临床表型，下颌肿大，牙周炎性反应；胫骨侧弯，骨皮质增厚，骨髓腔变窄，骨小梁的厚度和数目减少；血清中ALP的含量增高。Ano5 C360Y 突变KI小鼠也同样重复出部分中国GDD患者的临床表型，但是与Ano5 KO小鼠稍有不同。组织学显示在下颌骨的下颌支部位有赘生物，类似中国GDD患者的牙骨质瘤表现。牙周部位存在明显的炎症反应，IL-6和TNF-a的表达增高。长骨的表型与Ano5 KO小鼠相似。.获得表型效应后，我们进行了机制探索。1. Ano5 KO小鼠的mCOB中矿化结节增多，ALP表达水平升高，骨形成和矿化相关因子（Ocn、Col1a1、Opn、Opg、Rankl、Runx2和Osterix等）的表达发生发生改变，结合上述体外实验结果，提示Ano5在成骨细胞分化的过程中发挥负调控作用。2. Ano5 KO小鼠BMMs破骨诱导后发，发现破骨细胞形成减少，骨吸收的能力降低。Ano5 C360Y 突变KI小鼠mCOB也存在成骨和矿化增强和破骨功能降低的现象。3.对Ano5 KO小鼠的胫骨组织进行转录组芯片筛查后，发现了有意义的mRNA和microRNA，主要涉及成骨分化和矿化，以及破骨细胞融合的相关信号通路。.本项目揭示ANO5基因突变导致GDD的可能发病机制，对于指导疾病早期干预，开发针对中国GDD患者的治疗靶提供可靠的实验依据。