LRP6基因突变及DNA甲基化导致先天缺牙表型差异的机制研究

The mutation of LRP6, the WNT co-receptor, has just been related with congenital tooth agenesis. While its genetical mechanism and clinical character remains unclear, we identified novel LRP6 mutations in Chinese patients with tooth agenesis, and noticed the ectodermal dysplasia appearance. To test the hypothesis that “LRP6 mutation causes tooth agenesis via WNT-EDA signal pathway”, we plan to use in vitro culture of tooth germ cell, Lrp6 knockout mice, organ culture of tooth germ to get multilevel evidence. It is also noticeable that patients from same family with same LRP6 mutation have different pattern of miss teeth. Given that other gene mutation and individual discrepancy have been ruled out, DNA methylation difference was screened between missing tooth site and normal control from the same patient. To test the hypothesis that “LRP6 mutation and DNA methylation of specific promoter contributed to tooth agenesis of certain dentition site”, we plan to use DNA methylation array for screening, and to artificially manipulate the DNA methylation status to pheno-copy or rescue the development defect in Lrp6+/- tooth germ. The results of this study will help us to further uncover the mechanism of tooth development and to provide new insight for the prevention and treatment of tooth agenesis.

编码WNT通路受体的LRP6基因是新发现的与先天缺牙相关的基因，其突变致病机制和临床特点尚不清楚。本研究以高通量测序在中国先天缺牙患者中发现了新的LRP6突变，并首次发现LRP6突变的患者有外胚层发育不全表现，提出科学假设“LRP6突变通过WNT信号通路影响EDA信号通路导致先天缺牙”，拟通过牙胚细胞培养、基因敲除动物、牙胚器官培养在体外、体内实验证实。.此外，在家系中携带同样LRP6突变的患者各自先天缺牙数目和缺牙位点分布差别大，在除外其他基因变异、个体组织差异后进行DNA甲基化状态筛查的基础上，首次提出“LRP6基因突变和启动子DNA甲基化共同导致先天缺牙”假设。拟通过甲基化芯片对比筛查，人工编辑模拟或纠正特定位点启动子DNA高甲基化状态，观察牙发育和成牙相关基因表达，证实LRP6杂合突变合并启动子DNA高甲基化对牙齿发育的影响。这将对认识、预防和治疗先天缺牙发育畸形提供新的思路。

本研究探索了先天缺牙患者LRP6基因变异的情况和临床表型。通过突变功能预测、蛋白构象分析、荧光素酶报告基因试验初步评估了突变的功能影响。.1，初步完成LRP6基因突变患者临床表型记录。.对6个LRP6基因突变的患者家系进行了X线检查，明确缺失牙情况，在一例先证者中发现了外胚叶发育不全的表型。在一个家系中发现多指表型。.2. 新发现了6个未见报道的LRP6基因变异（c.2292G>A, c.195dup, c.1095dup, c.1681C>T，c.2840T>C，c.1154G>C）。对发现LRP6基因突变的患者进行了sanger测序验证以及家族共分离分析。通过遗传分析，确认了发现的LRP6突变的致病性。.3. 对发现的LRP6基因突变进行了功能分析.首选汇总了发现LRP6基因突变在多肽链上的具体位置，其次预测了发现的移码和无义突变对蛋白三维结构的影响，最后，通过构建野生型质粒和突变质粒，体外转染后荧光素酶报告基因实验，验证了发现的LRP6突变影响下游WNT通路的活力。.4. 初步了解了LRP6基因在小鼠牙胚发育中的时空表达模式。E11.5，在牙胚开始发育前，LRP6表达于上皮和间充质组织，E12.5-13.5，在蕾状期，LRP6主要表达欲牙胚上皮组织。E14.5帽状期，牙胚上皮和间充质都有LRP6表达，其中间充质部分的表达更强。E15.5-16.5钟状期，LRP6主要表达在牙乳头间充质和成釉器的内釉上皮部分。.5. 初步了解了LRP6基因变异患者缺牙的位置分布特征。对迄今文献报道的39例LRP6基因变异先天缺牙患者缺失牙位统计分析，发现上颌侧切牙为最常见的缺牙位点。

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