汉族家族性脑海绵状血管畸形CCM1/KRIT1基因新突变的功能验证及mRNA异常剪接的机制研究

Familial cerebral cavernous malformation (FCCM) is an autosomal-dominant disease with incomplete penetrance, clinically manifesting as hemorrhage, epilepsy and neurological deficits. The pathogenic genes of FCCM have been located in three chromosomal fragments: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10; nevertheless, the mutations exhibit considerable heterogeneity and there is a paucity of relevant studies in the Chinese population. In our previous work, we collected a large-sample Chinese FCCM families and reported several new mutations, including nonsense, deletion and splicing-acceptor-site variants. All these mutations are located in the CCM1/KRIT1 gene, while the effects of these mutations on the function of KRIT1 protein remain unclear. Moreover, bioinformatic analyses indicate that these mutations can not only affect the coding potential, but leading to aberrant splicing as well. The underlying mechanisms include abnormal splicing site recognition and abnormal splicing regulation, which may cause exon skipping. In this study, we intend to investigate the mechanism of mutation-related aberrant splicing by using minigene splicing reporting system and RNA pull-down methods. Then, we will construct an in-vitro eukaryotic expression system, in which we can validate the effects of mutations on the function of KRIT1 protein. This study is expected to provide crucial evidence for the pathogenic mechanism and genetic diagnosis of FCCM.

家族性脑海绵状血管畸形（FCCM）是一种具有不完全外显率的常染色体显性遗传病，可导致出血、癫痫或神经功能缺损。目前FCCM的致病基因定位于CCM1/KRIT1、CCM2/MGC4607、CCM3/PDCD10三个片段，但突变位点具有显著异质性且汉族人群研究较少。前期工作中，我们收集了大宗汉族FCCM家系，报道了多个新的无义、缺失、剪接受体位点突变，均位于CCM1/KRIT1基因上，但这些突变对KRIT1蛋白功能的影响尚不明确。而且，生物信息学分析提示这些突变除影响编码潜能之外，还可能影响mRNA的剪接，包括剪接位点识别异常和剪接调控异常，从而导致外显子跳跃。本研究首先利用minigene剪接报告系统和RNA pull-down技术探讨这些突变导致mRNA异常剪接的机制，进而构建体外真核表达体系，在细胞水平验证这些突变对KRIT1蛋白功能的影响，将为FCCM的致病机制和基因诊断提供重要依据。

家族性脑海绵状血管畸形（FCCM）是一种具有不完全外显率的常染色体显性遗传病，前期本课题组通过大宗汉族FCCM家系研究发现了多个位于CCM1/KRIT1基因上的新突变，包含无义、缺失、剪接受体位点突变，但这些基因座变异的功能影响及致病性尚不明确。生物信息学分析提示这些突变除影响编码潜能之外，还可能影响mRNA的剪接，包括剪接位点识别异常和剪接调控异常，从而导致外显子跳跃。.本研究的第一部分通过构建CCM1/KRIT1基因突变的minigene剪接报告系统分析mRNA的剪接异常，发现c.1412-1G>A突变会造成13号内含子滞留16bp，mRNA产物为c.1412-1_1412-16insGAATGATGTTTTTCAA，而其他外显子突变c.1864C>T、c.1980A>G、c.1197_1200delCAAA未影响mRNA剪接。.本研究的第二部分通过构建CCM1/KRIT1基因点突变的体外真核表达体系进行功能验证，证实点突变所导致的截短KRIT1蛋白不能正常进行核转运，同时影响ICAP1α蛋白的核转运。基因突变还促进血管内皮细胞的血管生成能力，显著增强血管内皮细胞的增殖与迁移功能，促进细胞周期从G0/G1期到S期进展，并抑制血管内皮细胞的凋亡。超微结构分析发现突变型血管内皮细胞含大量空泡及膨大内质网、线粒体、胞饮囊泡，代谢更活跃。.本研究的第三部分利用CRISPR/Cas9技术构建CCM1/KRIT1基因点突变细胞模型与哺乳动物（小鼠）模型，发现纯合KRIT1-null突变体在胚胎发育的早期阶段致死，证实了基因突变致病的二次打击机制。.本研究不仅证实了CCM1/KRIT1基因突变的功能影响及致病性，为FCCM的致病机制和基因诊断提供重要依据，还构建了点突变细胞与动物模型，为进一步开发基因治疗手段奠定关键基础。