成骨不全症新型二代捕获测序平台的建立及准确校正致病基因突变的分子靶向治疗探索

Osteogenesis imperfecta（OI）is the extremely severe and the most common monogenic heritable bone disease with phenotypes of osteoporosis, reduced bone strength, repeated fractures, severe deformity and dysfunction of bone. The diagnosis and treatment of OI is backward in China. The pathogenesis is unclear, the effective treatment is unavailable. We will construct a new targeted next-generation sequencing (NGS) platform, in order to acquire the gene mutation information in a larger sample of OI patients. We detect the serum levels of new biochemical index, such as pigment epithelium derived factor, transforming growth factor beta and sclerostin, as well as the expression level of their encoding gene in bone. We detect bone mineral density, bone strength and skeletal microstructure parameters using high-resolution peripheral quantitative CT instrument, the concave test and three-point bending test. We deeply analyze the relationship between the gene mutations of OI and new information of phenotype. According to novel mutations of Chinese patients with OI, we will establish two kinds of transgenic mouse model of OI with IFITM5 c.184G>A mutation, and with SERPINF1 c.119C>T mutation. We will investigate the metabolism process of type I collagen and the phenotype of transgenic mouse models of OI. Mouse mesenchymal stem cells (MSCs) will be isolated and cultured. We precisely correct SERPINF1 c.184G>A and IFITM5 c.119C>T mutation of MSCs using the new CRISPR/Cas9 gene editing technique and then induce MSCs differentiating to osteoblasts. The induced MSCs are infused to OI transgenic mice. We evaluate the effects of gene edited MSCs on OI through observing the expression level of type I collagen and its regulating protein, the serum levels of pigment epithelium derived factor, transforming growth factor beta and sclerostin, the changes of bone microstructure, bone mineral density and bone strength. We will improve the diagnosis of OI through the targeted NGS platform, and elucidate the OI pathogenesis using new transgenic mice model, and establish a new treatment strategy through new gene editing technology.

成骨不全症是以骨质疏松为表型、危害极其严重的最常见单基因遗传性骨病，导致反复骨折、严重骨畸形及活动障碍，我国该病诊治水平落后。本研究构建新型二代捕获测序平台，获得大样本成骨不全患者基因突变信息。检测多种新型特异性生化指标及其编码基因表达水平，采用高分辨外周定量CT仪检测骨密度及骨微结构，获得成骨不全症的表型新信息，深入分析基因突变型与表型的关系。针对致病基因新突变，构建SERPINF1基因c.184G>A突变、IFITM5基因 c.119C>T突变新型小鼠模型，观察小鼠表型，探索成骨不全症发病机制。采用新型CRISPR/Cas9基因编辑技术，准确校正小鼠间充质干细胞SERPINF1基因、IFITM5基因的突变序列，观察基因编辑后干细胞对成骨不全小鼠I型胶原表达及调控的影响，小鼠骨密度、骨强度及骨微结构的变化，评估基因编辑后干细胞的分子靶向治疗作用，建立成骨不全症的治疗新策略。

成骨不全症（Osteogenesis imperfecta，OI）是最常见单基因遗传性骨病，以骨量减少、骨脆性增加、反复骨折和进行性骨骼畸形为主要特征，具有高致病性及致畸性，危害极其严重，但目前疾病的诊断及治疗面临严峻挑战，缺乏有效策略。本课题组率先建立了OI全国最大研究队列，为绘制中国人群的此类疾病基因突变谱和表型图谱打下坚实基础。自主设计构建包括700多种与骨骼疾病相关致病基因突变检测的基因芯片，覆盖目前已知的20种OI的致病基因，采用先进的外显子捕获二代测序平台，对380例OI患者家系进行了表型和致病基因突变谱的细致研究，共检出13种OI的致病基因突变，发现该平台对OI致病基因突变的检出率为91.8%，准确性为100%。结果显示，常染色体显性遗传I型胶原编码基因COL1A1和COL1A2是中国OI主要的致病基因（占77.9%），其次是常染色体显性遗传IFITM5基因（占4.5%），常染色体隐性遗传基因占8.4%，以SERPINF1、WNT1和FKBP10基因为主，揭示了我国OI患者独特的遗传学特点。为更加精确反映患者临床表型轻重程度，首次对我国OI患者临床表型进行赋值评分，并对基因型-表型关系进行定量分析，结果发现OI患者基因型与表型间存在关联，常染色体隐性遗传比显性遗传患者病情重，突变导致胶原结构改变者较数量减少者表型严重，揭示中国OI患者基因型-表型存在相关性。采用血清色素衍生上皮因子（PEDF）、硬骨抑素（SOST）等新型生化标志物，细致描绘了多种遗传性骨骼疾病的表型谱，发现PEDF是VI型OI的独特生化标记物，提高了疾病的诊疗水平。在国内最早探索多种双膦酸盐制剂治疗OI的疗效与安全性，发现其能够增加患者骨密度、降低骨折率。通过定量测量双膦酸盐治疗前后OI患儿椎体形态，发现唑来膦酸能够使OI患儿压缩椎体出现再塑形，改变了OI为不治之症的误区。此外，本课题组还构建特异性新型致病基因突变的OI大鼠模型，以深入揭示OI发病机制，本组还作为主要指笔者，发布了成骨不全症临床诊治指南，研究结果加深了对OI的致病机制的认识，为实现OI的精准诊断提供参考依据，提高OI诊治水平。