家族性并指（趾）多指（趾）畸形致病基因鉴定和分子机理研究

项目名称： 家族性并指（趾）多指（趾）畸形致病基因鉴定和分子机理研究

项目编号： No.81501847

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 杜野

作者单位： 北京大学

项目金额： 18万元

中文摘要： 家族性多指（趾）并指（趾）畸形（SPD）是常见单基因显性遗传病，严重影响患者生活质量。本项目组收集到一个SPD大家系，共6代94人，现存患者22人。采用外显子组测序技术确定了172个候选致病基因，包含140个单核苷酸位点突变和32个插入缺失突变。本研究将采用PCR-Sanger测序技术，在家系其他成员中对候选突变进一步验证，确定致病突变及相关基因；建立该突变基因鸡胚过表达模型，分析肢体发育表型，确定基因致病性；通过绿色荧光蛋白融合表达和激光共聚焦显微镜技术，研究基因在鸡胚发育过程中表达特征，确定突变对于其表达和定位的影响；在过表达野生型和突变型HOXD13鸡胚中，采用原位杂交方法检测致病基因转录水平，探讨二者调控关系及致病基因调控通路；采用生物信息学和生物化学相关方法，研究该突变对于蛋白质结构和功能的影响。本项目研究结果将有助于进一步阐明SPD发生机制，为疾病诊断和预防提供科学依据。

中文关键词： 遗传性疾病；致病基因；基因突变；外显子组测序；分子机制

英文摘要： Synpolydactyly (SPD), a common monogenic dominant disorder, has deleterious effects on the quality of life. In our previous study, we described a six-generation SPD family with 94 members and 22 affected members alive. Based on exome sequencing technology, 172 candidate causative genes bearing 140 single nucleotide variants and 32 insertion and deletion mutations were identified. To determine the causative mutation and related gene, we will screen these candidate mutations in other members of this family using PCR-Sanger sequencing method. By over expressing the mutated gene in developing chick limb and analyzing the resulting phenotype, we will further verify the pathogenicity of gene mutation. With green fluorescent protein fusion expression system and confocal laser scanning microscope technology, we will monitor the gene expression dynamics in limb development and reveal the effect of pathogenic mutation on gene expression and location. HOXD13 was demonstrated to be key regulator in limb development. Using in-situ hybridization technique to detect the difference in transcription of the causative gene between chick limbs over expressing wide type and mutated HOXD13, we will explore their regulatory relationship and then regulatory pathway. With related methods in informatics and biochemistry we will also study the effect of mutation on protein structure or function. This project will shed new light on understanding the molecular mechanism of SPD and provide scientific basis for diagnosis and prevention.

英文关键词： genetic disease;causative gene;gene mutation;exome sequencing;molecular mechanism