外胚层发育不良遗传性耳聋-甲发育不全综合征动物模型的建立及致病机制研究

The deafness and onychodystrohy syndrome (DDOD) is a kind of hereditary ectodermal dysplasia disease which affects patients'cognition development and work capacity. DDOD shows autosomal dominant inheritance of congenital deafness and onychodystrophy. Conical, hypoplastic teeth are also a feature. We have identified a mutation c.1516C>T (p.R506X) in ATP6V1B2 gene in three Chinese DDOD pedigrees through the whole human exome sequencing and bioinformatics technique. This ATP6V1B2 c.1516 C>T (p.R506X) mutation was absent in the parents, indicating the de novo nature of the mutation. The newly identified gene,ATP6V1B2, encodes V-ATPase,a H+ transporter.It expresses widely in tissues developing from ectoderm. Our former results show it expresses in the mouse cochlea,especially in organ of Corti and spiral ganglian neurons.Preliminary in vitro experiments in HEK293 cells showed that the ATP6V1B2 p.R506X causes PH abnormity in lysosome which further lead to the disorders of cell digestion and cell injury. All the above results show ATP6V1B2 gene deficiency correlates with DDOD.In this study, we will investigate on the function of the newly identified deafness gene responsible for DDOD syndrome by making a conditional cochlea Atp6v1b2 Knockdown mouse model and an Atp6v1b2 c.1516 C>T (p.R506X) mutation Knockin mouse model. Systemic phenotype,pathophysiology and biochemistry study will then be performed on the models. The results of this study will reveal the pathogenesis and molecular pathway of DDOD syndrome,help deepen the understanding of DDOD and provide basis for the biological treatment on DDOD.In addition, this study will underlie the development of preimplantation diagnosis and prenatal diagnosis for DDOD.

外胚层发育不良常染色体显性遗传耳聋-甲发育不全综合征(dominant deafness and onychodystrohy syndrome，DDOD)病因不明，表现为先天性重度感音神经性耳聋、甲发育不全或缺失、指骨融合或增多、牙齿畸形或缺失，严重影响患者的认知和劳动能力。我们前期利用外显子组测序技术在三个中国DDOD家系检测到ATP6V1B2基因新生突变p.R506X，发现该基因编码的H+转运蛋白V-ATPase在小鼠内耳毛细胞及神经元高表达，体外细胞模型证实该突变导致溶酶体酸度异常、细胞消化功能紊乱进而细胞损伤。上述结果显示ATP6V1B2基因缺陷与DDOD发病密切相关。本课题拟构建Atp6v1b2基因耳蜗选择性Knockdown小鼠模型及突变Knockin小鼠模型,对动物模型进行表型鉴定及在体病理、电生理、生化研究，明确DDOD的致病机制。研究结果将为该病的生物学治疗奠定基础。

显性遗传耳聋-甲发育不全（DDOD）综合征罕见，全球迄今共报道11个家系，3个来自申请人课题组。该病不仅引起耳聋，还表现为指甲、趾甲、指骨发育不良，严重影响患者认知及劳动能力。本课题组前期利用全外显子组测序及生物信息学技术率先鉴定了其致病基因ATP6V1B2，发现其新生无义突变c.1516 C>T（p.Arg506X）与DDOD表型密切关联。该基因编码V-ATPase，在哺乳动物内耳、脑、心脏、肾等多系统表达，knockout小鼠无法存活，基因功能研究甚少。在本项目资助下，课题组建立了鼠内耳中阶局部注射Morpholino敲低基因表达的研究方法，发现耳蜗局部Atp6v1b2基因knockdown后小鼠发生重度耳聋，证实了该基因与听觉相关。进一步体外细胞功能分析发现c.1516C>T是单倍剂量不足突变，导致V-ATPase水解和离子转运活性减低、细胞溶酶体酸度下降，从而提出了“溶酶体降解功能不全导致的细胞代谢障碍是DDOD综合征关键病因”的学术观点。上述工作发表在Cell Research杂志，被OMIM数据库收录和引用。 “ATP6V1B2基因突变体及其应用”获得国家发明专利授权。课题执行过程中，课题组收集到第四个中国DDOD家系，该家系有患者两名，测序证实致病突变仍为ATP6V1B2 c.1516C>T。.对四个DDOD家系进行深入表型分析，发现患者人工耳蜗植入后言语康复效果欠佳，在明确人工耳蜗能补偿患者的听觉的前提下，考虑言语康复问题与中枢有关。为进一步研究DDOD致病机制，课题组利用基因打靶技术建立了ATP6V1B2 c.1516C>T突变knockin小鼠模型，表型分析显示该knockin小鼠模型表现出听力正常、学习记忆障碍。针对小鼠的学习记忆障碍进行功能性磁共振脑扫描和组织学研究，结果提示：模型小鼠脑实质体积减小，左侧颞叶、枕叶以及小脑灰质密度降低；小鼠模型脑功能网络出现广泛异常：主要涉及边缘系统的多个脑区，部分脑区间的功能连接出现代偿性增强；纯合子小鼠海马组织Atp6v1b2蛋白表达降低、神经元个数显著降低，影响海马功能。关于小鼠模型没有产生耳聋表型，初步考虑可能在耳蜗存在某种补偿机制，目前在针对小鼠耳蜗、脑组织分别进行RNA-seq，结果在分析中。