拷贝数变异诱发外耳发育畸形的分子机制研究

External ear is one of the most important sensor organs derived from the first and second branchial arches, which are composed of neural crest cells and mesenchymal cells. External ear malformation, also known as microtia, significantly impairs the physical and metal healthy of the patients. Recent studies have been focused on the causal mutation discovery of the disease, however, no causal mutation has been reported till now. Our team has identified the first causal mutation, i.e., a novel copy number variation (CNV), for the disease in three large pedigrees using genome-wide linkage analysis and subsequent target-capture sequencing. The copy number variation is located at the regulatory region of the HMX1 gene and harbors a “conserved non-coding element (CNE)”, which is predicted as an enhancer for HMX1 in natural animal models. To demonstrate the effects of this CNV on the CNE and the expression level of HMX1, a chromosome conformation capture sequencing in neural crest stem cells will be performed. To disclose the biological mechanism of external ear malformation induced by the CNV, gene edit mice of the orthologue sequence of the CNV will be constructed using CRISPR-Cas9 system. Our results will provide genetic evidence for clinical therapy and prevention of external ear malformation, which can be used for animal breeding to generate economic efficiency.

外耳主要是由神经嵴细胞发育而来的人体重要感知器官，外耳畸形严重影响患者身心健康。揭示外耳畸形的遗传风险因素是目前的研究热点，但至今仍无人类非综合征型外耳畸形的致病突变被报道。本研究团队通过连锁定位和目标区域捕获测序手段在我国三个非综合征型外耳畸形大家系中探测到一个新发的拷贝数变异，第一次发现了人类外耳畸形的致病突变。该拷贝数变异位于HMX1基因调控区并包含一个“保守非编码元件”；天然动物模型中发现该元件可能是调控HMX1基因表达的增强子。为了探查该拷贝数变异如何影响“保守非编码元件”对HMX1基因表达的调控，我们将采用染色体构象捕获测序的方法在人类神经嵴干细胞中对该突变进行研究。同时还将使用CRISPR-Cas9技术在小鼠中对该突变的同源序列进行基因编辑，以揭示该突变导致外耳畸形的生物学机制。本研究结果将为外耳畸形的治疗和预防提供遗传学基础，也可应用于动物育种中产生直接经济效益。

本团队前期研究中发现了一个位于HMX1基因调控区并包含“保守非编码元件”拷贝数变异，该拷贝数变异是第一个发现的人类外耳畸形致病突变。在前期基础下，本项目成功将hiPSCs诱导分化为hNCCs，并且通过对人源诱导的神经嵴细胞（hNCCs）的表观遗传学模式分析（p300，H3K27ac，H3K4me1，ATAC-seq），发现在三个家系的重叠区域内存在一个候选增强子。针对候选增强子，我们采用CRISPR-Cas9技术在小鼠中对该突变的同源序列进行基因编辑，成功构建了该拷贝数变异的基因编辑小鼠，并验证了该模型的有效性。同时，通过对不同发育阶段突变体小鼠外耳cDNA 文库构建和real-time PCR检测，我们探索了该突变导致外耳畸形的相关生物学机制，并最终定位了包括HMX1在内的8个重要候选致病基因及5个相关信号通路，将可揭示我国非综合征型外耳畸形的病因。