结节性硬化症中TSC1/TSC2基因内含子突变的致病机制及临床意义研究

Approximately twenty percent of patients with tuberous sclerosis complex could not identify pathogenic mutations in the TSC1 or TSC2 genes. The unknown etiology has confused the application of mTOR inhibitors in these patients. Recent advances in next-generation sequencing technologies have enabled the detection of numerous intronic mutations, which play an emergent role in diverse diseases. In the previous study, we assessed 83 families of tuberous sclerosis complex by next-generation sequencing and found 13 patients who did not detect pathogenic mutations. In the preliminary experiment, we found five novel TSC1 or TSC2 gene intronic mutations co-segregated with tuberous sclerosis complex by deep-analyzing the next-generation sequencing data and validated by Sanger sequencing. Our current works showed that two intronic mutations resulted in abnormal splicing of the corresponding mRNA by constructing a minigene vector. However, it is not clear that the effects of TSC1 or TSC2 intronic mutations on the function of the corresponding protein and its pathogenesis in tuberous sclerosis complex. In this proposed project, we plan to expand our cohort of TSC1 or TSC2 intronic mutations cases of Chinese population. We will analysis the abnormal splicing and protein function by constructing minigene vector and CRISPR/Cas9 system in vitro assays. In addition, we will study the effect of mTOR inhibitors in vitro cell model. This study will enhance our understandings of functional roles of TSC1 and TSC2 intronic mutations in the pathogenesis of tuberous sclerosis complex and provide useful information for proper application of mTOR inhibitors.

约20%结节性硬化症（TSC）患者不能检出TSC1或TSC2基因的致病突变，致病机制不明给临床mTOR抑制剂的应用带来困惑，最新研究提示基因内含子突变在疾病中发挥重要作用。前期我们对83个TSC家系进行二代测序基因检测，其中13例患者未检出致病突变。我们对二代测序数据深入分析，并应用Sanger测序对患者及家系成员进行验证，新发现5种TSC1或TSC2基因内含子突变与疾病共分离。通过构建微小基因初步验证其中2种内含子突变可导致相应mRNA发生异常剪接，但是对相应蛋白质功能的影响及致病机制尚未明确。本项目拟扩大收录TSC1或TSC2基因内含子突变家系，应用微小基因载体和CRISPR/Cas9系统分别构建mRNA异常剪接和蛋白质功能体外分析模型，明确mTOR抑制剂对细胞模型的影响。本研究将阐明TSC1和TSC2基因内含子突变导致TSC的致病机制，为mTOR抑制剂的个体化用药奠定理论依据。

约20%结节性硬化症（TSC）患者不能检出TSC1或TSC2基因的致病突变，致病机制不明给临床mTOR抑制剂的应用带来困惑，最新研究提示基因内含子突变在疾病中发挥重要作用。在项目执行期间，我们对170个中国结节性硬化症家系进行了二代测序基因检测，新发现17个TSC1或TSC2基因突变位点，检测出并鉴定了12种内含子突变。我们初步绘制了中国结节性硬化症相关肾脏血管平滑肌脂肪瘤患者基因型图谱，阐明TSC1和TSC2基因内含子突变的致病机制，为mTOR抑制剂的个体化用药奠定理论依据。我们在多中心回顾性分析了mTOR抑制剂依维莫司与西罗莫司对比治疗结节性硬化症相关肾脏血管平滑肌脂肪瘤（TSC-AML）的可行性及安全性，进一步分析了mTOR抑制剂与基因型的相关性。此外，结合我国国情我们进一步探讨了mTOR抑制剂全剂量诱导联合低剂量维持序贯治疗TSC-AML的个体化治疗新模式，一方面减少了mTOR抑制剂治疗带来的副作用，另一方面也大大降低了患者的经济负担，具有重要的理论价值和社会经济意义。