I型牙本质发育不良新致病基因的分子机制及功能研究

Dentin dysplasia type I(DD-1) is a rare autosomal dominant hereditary disorder which serioulsly affects the root development of teeth among teenagers causing spontaneous teeth falling.The etiology is still unknown.In this research,we have located a novel mutation on 18 Chromosome in Chinese family patients through whole genome-wide SNP array and STR linkage analysis. Further exploring by the target sequence capture sequencing, bioinformatics and co-segregation analyses clone the pathogenic mutation associated with DD-I. We will follow up the family to collect more dropped teeth for understanding teeth ultramicroscopic structure and major elements. The causal relationship between the mutation and DD-I approve by the application of RT-PCR and nucleside transporter vector. Furthermore the hypothesis concerned with the novel genes responsible for DD-I will be examed by means of a series of experiment of zebrafish model,observaton of zebrafish teeth phenotype and inhibition of zebrafish homologous protein from the mutation.We can make a further study on the molecular mechanism of this pathogenetic gene during tooth root development and clarify the clone of DD-1 pathogenetic gene and its molecular pathogenetic mechanism.This research will complement theoretical knowledges on the mechanism of physiologic dentinogenesis and provide a scientific basis for the diagnosis and treatment of DD-1.

遗传性I型牙本质发育不良（DD-I）是一类严重影响牙齿根部发育而导致青少年时期牙齿自发脱落的常染色体显性遗传病，目前对其致病机理仍不明了。本课题组采用SNP芯片全基因组扫描和STR分析将我们收集到的DD-I家系致病基因定位到了18号染色体一个新突变位点上，并结合区段捕获测序和家系内共分离克隆到了该致病基因，进一步明确DD-I的新致病基因的存在。本研究将对脱落患牙进行超微结构和能谱分析，同时，通过RT-PCR及载体表达等技术手段提示新的致病基因与DD-Ⅰ发生的关系；建立斑马鱼相关模型，通过抑制斑马鱼致病基因同源基因蛋白的表达，观察斑马鱼牙齿表型变化，确定新的致病基因功能等实验，进一步证实我们提出的该致病基因影响DD-Ⅰ牙本质发育的假说，为阐明DD-I疾病致病基因及该基因在牙根的生理发育机制功能方面补充新的理论知识，为DD-I疾病的临床诊治提供新的遗传学基础。

I 型遗传性牙本质发育不良（DD-I）是一类严重影响牙齿根部发育而导致患者牙齿自发脱落的常染色体显性遗传病，目前对其致病机理仍不明了。为了阐述该病的发病机理我们设计了该课题：通过一个含有10个DD-I患者的大家系，我们将获选基因定位在18号染色体18q21.2-q21.33之间的9.63 Mb范围内，通过家系共分离策略发现家系中的10名患者的 VPS4B基因的7号内含子发生IVS7+46C>G 突变。从患者的牙龈成纤维细胞分离到了插入45bp 的异常转录本。蛋白结构预测发现15个氨基酸的插入改变了VPS4B的ATP结合盒的结构。突变明显改变了VPS4B的mRNA和蛋白质的表达及基因的亚细胞定位，预示了VPS4B功能的丢失。通过牙龈成纤维细胞分析发现VPS4B作为Wnt/beta-catenin 信号的上游调控信号分子调控牙本质的形成。此外 vps4b敲低斑马鱼显示出DD-I类似的症状，如牙齿数目减少或缺失，并且通过人野生型VPS4B mRNA的拯救突变表型减轻。我们还观察到VPS4B的低表达负向调控牙本质形成相关基因的表达。科学意义：本课题的完成对遗传性 DD-I 的致病基因的定位克隆和分子发病机制研究揭开新的篇章，充分揭示 DD-I 的分子致病机理，完成牙本质发育缺陷性疾病致病基因寻找的最后一个盲区，为牙齿尤其是牙本质和牙根的生理发育机制提供新的理论知识和研究线索，为 DD-I 型的临床诊断、治疗、遗传咨询和预防提供实验和理论依据。