基于多重PCR-HRM的高通量视网膜色素变性基因突变筛查与RP新致病基因鉴定

Retinitis pigmentosa (RP) is the most common and severe hereditary ocular disease, leading to severe visual impairments or even blindness. RP presents significant and complex clinical and genetic heterogeneities involvinging massive disease-causing genes. Therefore, it has been a great challenge for molecular diagnosis of RP. In addition, a large number of unknown RP-causing genes are yet to be discovered. In this project, we will continue to collect blood samples and clinical data of RP disease to expand the RP genetic resources database based on our previous work. We will develop a high throughput and cost-effective massive mutation screening technical platform for detecting RP-causing genes based on PCR-HRM (High-resolution melting) analysis with DNA pooling as PCR templates. And then we will identify the causative mutations of known RP-causing genes in a larger number of patients with RP disease, and construct the distribution of genes and their mutation spectra in Chinese population of RP patients. Later, we will discover some new disease-causing genes using the genome-wide SNP scans, linkage analysis and targeted next-generation sequencing or exome sequencing technology in the RP families, which have been excluded the known RP genes. Furthermore, we will study on the biological function of the new RP-causing genes for illuminating their pathogenic mechanisms. In conclusion, if the project has been implemented and completed successfully, we will achieve clinical molecular genetics diagnosis for RP, and offer the scientific strategies for gene therapy, prevention and intervention of the RP disease. The discovery of the new RP-causing genes will further enrich and expand our understanding the RP disease.

视网膜色素变性(RP)是一类常见且危害最严重的遗传性致盲眼病，具临床表现复杂，遗传异质性高和致病基因众多等特征，使其临床基因诊断颇具挑战性。另外，尚有相当数量的未知RP致病基因亟待挖掘。本课题拟在前期基础上扩容RP遗传资源专题数据库，应用基于DNA池的PCR-HRM分析技术，建立高性价比和高通量的RP致病基因突变筛查技术平台，大量筛查和鉴定RP致病基因突变，构建我国RP致病基因图谱及其突变频谱。对于排除已知致病基因的RP家系，则应用全基因组SNP扫描、连锁分析和目标序列捕获的二代测序或外显子组测序等技术发现新致病基因，分析其生物学功能，阐明其致病的分子机制。项目的顺利实施和完成可望实现RP临床分子诊断，为该类疾病的基因治疗、预防与干预决策制定提供科学依据；RP新致病基因的发现亦将进一步丰富和拓展人类对RP的认知。

视网膜色素变性(RP)是一类常见且危害最严重的遗传性致盲眼病，具临床表现复杂，遗传异质性高和致病基因众多等特征。目前，基因治疗可能是根治RP潜在的唯一手段，而基因治疗的前提是筛查和鉴定RP患者的致病基因。本项目已完成如下内容：1) 基于文献数据挖掘构建中国人群视网膜色素变性(RP)致病基因频谱，结果显示在507个RP相关性疾病家系或先证者中涉及67个RP致病基因，其中12个致病基因在10个以上家系或先证者中发现，占总家系或先证者的76.5%；31个致病基因仅在一个家系或先证者中存在，占总致病基因的46.3%。2) 建立基于gDNA池的多重PCR-HRM 分析技术的高通量RP致病基因突变筛查平台，结果在项目实施过程中发现HRM曲线异常的常常为核苷酸多态性，筛查和验证工作量和费用剧增，与预期出入太大。随着目标捕获二代测序技术和全外显子组测序技术的发展，其费用大幅降低。基于此，为适应新技术的发展和兼顾效益，我们及时调整研究策略，即采用全外显子组测序技术筛查RP致病基因。3) RP相关疾病家系的致病基因筛查和鉴定：我们在176个RP相关疾病家系中筛查和鉴定了其中20个家系的致病基因及其突变，尚有36个家系正在进行全外显子组测序分析。已鉴定11个RP致病基因(RHO、RLBP1、TULP1、PCDH15、RP1、RP2、BEST1、CRX、CRB1、MFRP和CHM)，其中14个为新致病性突变位点。 4) 其他遗传性眼疾病致病基因筛查和鉴定：受本项目资助，我们还筛查和鉴定了60个其他遗传性眼疾病家系或散发病例的致病基因及其突变，包括HPS综合征(眼底白化)、眼外肌纤维化、先天性白内障、原发性开角青光眼和LHON。 5) 目前以上研究结果部分已发表学术论文5篇，获授权国家发明专利1项，已撰写学术论文3篇。总之，本项目丰富和拓展人类对RP等遗传性眼病的认知，为该类疾病的诊疗与干预决策制定提供科学依据。