DLX3基因突变诱导TDO综合征的分子机制及利用CRISPR-Cas9技术基因纠正治疗研究

Mutation of the distal-less gene (DLX3) is closely associated with Tricho-Dento-Osseous syndrome (TDO), which is an inherited autosomal dominant disorder and mainly characterized by kinky hair, dentin hypoplasia, and increased thickness and density of cranial bones. So far, although DLX3 is proved to be essential for the development of hair, teeth and cranial bones, yet the underlying mechanism how DLX3 mutation leads to TDO syndrome remains largely unknown. Our study group identifies a novel DLX3 mutation (c.533A>G, Q178R) in the world, and has demonstrated that this mutation not only can influence the osteogenic potential of bone marrow mesenchymal stem cells (BMSCs), but also alters the senescence process. We, for the first time, propose the hypothesis that DLX3 can affect the hair, teeth and bone development though regulating aging process. We plan to cultivate the BMSCs from TDO patients and normal healthy donors and take these as the experimental materials to screen the genes regulated by DLX3 on the level of DNA methylation, Lnc RNA and transcriptome through high-throughput technology. To explore the regulation mechanism of DLX3 in hair, teeth and bone formation and development, we will construct and yield the lentivirus-carrying wild type DLX3, mutant DLX3 and shDLX3, respectively, then use then to validate and evaluate the differential gene candidates found in the high—throughput data. Next, we will establish wild type DLX3 and mutant DLX3 transgenic mice to create TDO mice model, and then to confirm the mechanism in vivo through tissue section and immunostaining technology. Finally, to try to treat the pathologic features of TDO syndrome, we will design a CRISPR-Cas9 system to correct the mutation site of DLX3 in the transgenic mice. If we achieve the favorable results, they will provide us a solid theory and practice foundation to try to treat TDO syndrome as well as other genetic diseases.

TDO综合征是罕见的外胚层发育不全的DLX3单基因突变导致的常染色体显性遗传病。头发、牙齿和骨发育异常是其临床表征。目前，DLX3突变导致TDO的分子机制仍不清晰。本课题组发现新的DLX3（Q178R）突变，并初步证明该突变可以影响成骨分化和功能，还能影响骨髓间质干细胞的衰老进程。首次提出“DLX3通过调控衰老进程来影响骨、毛发及牙齿发育的科学假设”。拟通过培养TDO病人和正常人的骨髓间充质干细胞，用高通量测序技术在甲基化、LncRNA和转录组水平，对DLX3调控基因进行筛选；用病毒介导的DLX3野生型和突变型基因，敲低DLX3表达的shRNA，验证差异明显的候选分子，探寻并验证调控机制；建立野生型和突变型DLX3转基因鼠模型，在体内验证并阐明DLX3在发育过程中的作用，利用CRISPR-Cas9系统精确纠正突变DLX3基因，尝试纠正小鼠疾病性状表现，为单基因遗传病的治疗提供理论基础。

本研究针对导致毛发-牙齿-骨（TDO）综合证的致病基因DLX3作用机制进行研究。计划确定患者突变类型、通过患者的骨髓间充质干细胞及牙髓干细胞研究DLX3如何通过“衰老”对成骨和成牙的作用、研究DLX3在甲基化、长链非编码RNA和转录组水平上的调控机制、并希望通过动物实验对突变基因纠正。研究基本按照计划进行，取得良好结果。.1.本研究发现该TDO患者DLX3第三外显子存在突变，即c.533A>G(p.Q178R)，这是中国人首次报道的新突变。.2.发现并验证DLX3基因对骨髓间充质干细胞成骨分化和衰老的正向调控。衰老减缓的骨髓间充质干细胞通过延长其细胞生命周期和产生更多的子代骨细胞，弥补细胞较差的成骨能力，从而导致骨密度增高的病理机制。构建过表达突变型DLX3(Q178R)转基因小鼠，发现突变的小鼠不仅表现出骨密度的明显增高，衰老的延缓，并且随着年龄的增长，还表现出可以减少因衰老引起的骨密度减少，进一步在体内验证了细胞实验结果。.3.首次报导长链非编码RNA H19（lncRNA）在TDO综合征骨发育中的作用。采用lncRNA基因芯片技术分析，发现了患者的骨髓间充质干细胞中lncRNA H19的表达水平明显降低而其甲基化水平明显增加。发现H19/miR-675会明显抑制骨髓间充质干细胞的增殖，而DLX3突变会明显抑制H19/miR-675的表达，因此DLX3突变通过抑制H19/miR-675的表达而达到促进患者骨髓间充质干细胞的增殖，从机理上解释了患者的骨密度增加。.4.首次发现DLX3－H19－DLX3环路在TDO综合征牙本质发育中的作用。lncRNA基因芯片结果提示H19在TDO综合征患者体内表现为低表达，高甲基化。机制研究结果显示，H19基因是通过SAHH降低DLX3的甲基化，促进DLX3的表达而达到促进牙髓干细胞成牙分化的。. 上述研究结果深入说明了疾病机理，同时对骨的发育调控提供了新的思路。

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