外胚层发育不良综合征相关新基因的发现与发病机理的初步研究

Ectoderm dysplasia (ED) syndrome is a kind of rare inheritance disease. Patients who suffering from ED usually manifest distinctive abnormal development of structures including the skin, hair, nails, teeth, and sweat glands, with the underlying mechanism yet incompletely understood. Previous studies have demonstrated that human hypohidrotic ED (HED) is primarily associated with mutations in EDA, EDAR, and EDARADD genes, which down-regulates the functionality RANK-RANKL/NF-κB pathways and lead to abnormal cell proliferation and differentiation that finally result in HED. However, it remains yet unclear whether there might exist other genetic factors that also contribute to human HED. To this end, we have systematically collected samples from clinically ascertained HED patients and subjected their DNA for high-throughput exome-sequencing. Interestingly, a common frame-shift mutation in WF1 gene was identified in many HED patients of no obvious blood descendants. Subsequently, we found that such WF1 gene mutation led to differentiation abnormality of the alveolar bone marrow mesenchynlal stem cells (Al-BMSCs), and genetic ablation of WF1 also drastically decreased the activity of NF-κB signaling pathway in HEK293 cells. Furthermore, through yeast two hybrid screening and later co-immunoprecipitation (Co-IP) analysis, RANK was identified as a potent interacting partner for WF1. This prompted us to hypothesize that WF1 mutation might impact on the NF-κB signaling through direct interaction with RANK, which finally lead to human HED. Here, we propose to test this hypothesis by first establishing the cellular and mouse models, which are either directly derived from patients or have the WF1 gene genetically manipulated; and taking multiple approaches, e.g. genetic screening, proteomic analysis and cell behavior and differentiation study and so on, to deepen our mechanistic understanding in this exciting new arena.

外胚层发育不良征(ED)是一类遗传罕见病，累及牙齿、颌骨、汗腺等多器官。基因突变导致的部分信号通路活性的改变是ED发生的主要原因。收集来源于临床已确诊的少汗型ED(HED) 病例的血液样本外显子高通量测序，我们在多个无血缘关系的病人中发现了此前从未被报道过 的WF1基因移码突变。初步研究表明此类突变会引起牙槽骨来源干细胞分化异常，WF1基因敲除 导致胞内NF-κB活性下降，WF1与RANK存在相互作用。据此，本课题提出假说：WF1基因突变可能引发RANK-RANKL信号通路异常而下调NF-κB活性，从而导致细胞分化异常而诱发ED。我们拟通过建立WF1敲除的细胞、小鼠模型，结合永生化细胞株，运用遗传筛选和蛋白质组学方法深入研究WF1突变影响RANK-RANKL等信号通路而下调NF-κB活性的分子机制，全面探讨WF1基因突变参与ED发生发展。

外胚层发育不良征（ED）是一类遗传罕见病，累及牙齿、颌骨、汗腺等多器官。通过收集来源于临床已确诊的少汗型ED（HED）病例的血液样本进行外显子高通量测序，我们在数个无血缘关系的病人中发现了此前从未被报道过的WF1基因移码突变。.在取得正常人及病人牙槽骨组织后，进行牙槽骨骨髓间充质干细胞（Al-BMSCs）的体外培养。免疫组化检测 WF1 蛋白表达定位，RT-PCR 及 WB 定量分析 WF1 基因表达水平，发现病人Al-BMSCs的WF1 基因表达水平表达水平降低。同时，诱导细胞定向分化，检测发现病人 Al-BMSCs 细胞的成骨、成表皮分化功能减弱，推测 WF1 基因突变可能诱发细胞分化异常从而导致 HED 发生。.同时，我们已构建并获得EDAR基因全身敲除小鼠，和表皮、间充质选择性敲除小鼠，并进行了相关研究。对小鼠的腹部、背部、耳后和尾部皮肤，以及股骨、下颌骨和尾骨样品进行固定和切片，行HE和免疫荧光染色；同时，对小鼠的股骨、下颌骨和尾骨样品行micro-CT检测。结果显示，EDAR全敲小鼠和表皮选择性敲除小鼠的皮肤厚度减小，汗腺及皮脂腺消失，EDAR表达量明显降低；EDAR全敲小鼠下颌骨第三磨牙未萌出，尾骨末端有一弯折，尾骨、股骨和下颌骨密度则与野生型小鼠无明显差异。取表皮选择性敲除小鼠表皮组织行RNA-seq分析，发现较同窝flox/flox小鼠， EDAR在mRNA水平上明显降低，RANK-RANKL信号通路活性下调，在动物水平证实了 WF1 基因突变下调RANK-RANKL 信号通路活性而导致 Nemo 参与的经典 NF-κB 信号通路异常，最后引起细胞周期异常和 HED 发生。.利用酵母双杂交和免疫共沉淀-质谱技术鉴定 EDAR 相互作用的蛋白，并完成体外验证，为进一步揭示 WF1 基因通过与 RANK 蛋白相互作用来调节 RANK-RANKL 通路活性而导致 HED 发生做好了基础。.本课题从一个新的角度揭示 HED 发生发展可能的机制；一方面对 WF1 基因在外胚层发育成牙齿、毛发等中的关键作用有了初步认识，同时也为我们全面深入认识 HED 发生发展的分子机理、为发展针对 HED 的新的靶向诊断治疗手段提供有力的研究基础和理论指导。