肿瘤相关基因CEACAM5和SETD6与脊髓血管畸形致病的关联性分析及功能研究

Spinal arteriovenous malformation (SAVM) is a complicated sort of neurological disease that causes severe disability in young adults, which is considered as a congenital disease as well as cerebral arteriovenous malformation. As a sporadic disease, there is no genetic study of this disease yet. Based on the whole exon sequencing and screening strategy of the core families, the de novo mutant genes CEACAM5 and SETD6 were found in our previous study. According to the literatures reported before, both of them were associated with tumor. The CEACAM5 has ability to induce angiogenesis and the SETD6 is an important regulator of cell antioxidation. The biological functions of CEACAM5 and SETD6 are also the key mechanisms for the development of vascular malformation. We thus speculate that the CEACAM5 and SETD6 may be associated with the onset of SAVM. In this study, a larger number of patients and paired control subjects will be sequenced to establish the association between the mutations of CEACAM5 and SETD6 and the SAVM. In addition, the endothelial cells will be primarily cultured from patients with the mutations of CEACAM5 and SETD6 as the in vitro experimental model. Using these cultured cells, the mechanism of CEACAM5 and SETD6 in regulating the pathogenesis of SAVM will be elucidated by determining the expression level of mRNA and protein, the endothelial cell proliferation, migration and VEGF reaction, the ROS and oxidative damage effect markers. The current investigation will fill the gaps between CEACAM5 and SETD6 mutation and SAVM, which provides insights into the mechanisms of SAVM for combating the disease.

脊髓血管畸形是导致青壮年严重残疾的一类神经系统疾病，与脑血管畸形一样被认为是一种先天性疾病，目前尚无其遗传背景的研究。我们前期研究中采用基于核心家系的全外显子测序和筛选策略，发现了新发突变基因CEACAM5和SETD6。迄今文献报道CEACAM5和SETD6均与肿瘤相关，前者可诱导血管新生，后者是细胞抗氧化应激的重要调节因子，这些也是血管畸形发生的关键机制。因此我们推测他们可能与脊髓血管畸形致病相关。本研究拟利用大样本的患者和健康对照队列对上述基因进行关联分析，以验证其与脊髓血管畸形的相关性；同时拟使用CEACAM5和SETD6突变患者的畸形团内皮细胞构建细胞模型，通过分析突变对mRNA和蛋白表达水平、内皮血管新生功能、抗氧化应激能力的影响，来阐明CEACAM5和SETD6介导脊髓血管畸形发生的机制，将为研究血管畸形提供新思路。

脑、脊髓血管畸形是指位于脑和脊髓内的异常血管构筑，好发于青壮年，是一类高致残性疾患，多数情况下在出生时就已经存在。目前关于血管畸形的研究表明，血管新生和炎症通路是导致血管畸形的主要致病机制，缺血缺氧和氧化应激反应也参与其中。但是目前关于脊髓血管畸形的遗传背景研究及潜在的信号传导通路，尚有待深入系统的探讨。本课题组基于前期的临床基础研究，通过生物信息学分析确立了两个突变基因：CEACAM5 和 SETD6，并推测该基因可能导致脊髓血管畸形的发生发展。本项目旨在通过大样本的散发脊髓血管畸形病例和健康人对照，以验证CEACAM5 和SETD6与脊髓血管畸形发病的关联性；在功能学上，我们利用体外培养的脊髓血管内皮细胞，测定CEACAM5 和 SETD6在不同人群（携带突变基因的脊髓血管畸形患者、不携带突变基因的散发血管畸形患者、正常人群）内皮细胞上的表达情况；最后利用体外培养的CEACAM5突变内皮细胞模型和SERD6突变内皮细胞模型，以探索突变对内皮细胞血管新生功能和抗氧化应激能力的影响。研究发现：生殖系突变与脑脊髓血管畸形发生发展的关联性可能较低，遂转化研究思路，转而研究肿瘤相关基因的体细胞突变与血管畸形发生间的关系，并取得了如下研究成果：.（1）肿瘤相关通路基因KRAS/BRAF体细胞突变在脑脊髓动静脉畸形的发生发展中发挥了核心作用，此外，我们还发现高达87.1%的脑和脊髓血管畸形患者病变中存在KRAS/BRAF体细胞突变，进一步分析病灶组织内的突变丰度后证实KRAS/BRAF体细胞突变在动静脉畸形发生中的始动作用。.（2）首次全面阐释了脊髓动静脉畸形的自然病史，明确了脊髓动静脉畸形患者的自然病史恶劣，在临床工作中需要积极干预。.（3）基于本课题组的研究成果，结合前人对遗传学血管畸形的临床相关研究，提出中枢神经系统动静脉畸形患者可以通过口服药物沙利度胺获益，并于2019年申请了相关国家发明专利。