基于多家系HVA新致病基因鉴定及遗传学机制研究

Familial vitreous amyloidosis (HVA) is a genetic disease caused by the abnormal deposition of amyloidosis in the vitreous cavity due to the mutation of the transthyretin.(TTR) gene. Can cause severe visual impairment in patients without effective treatment. Therefore, it is of great significance to further explore the pathogenesis of HVA and find new therapeutic targets. Preliminary studies confirmed that TTR Gly83Arg mutation was associated with the incidence of HVA. Both the TTR Gly83Arg mutation and TTR c.-743A>T mutation could lead to the decrease of TTR protein expression in the blood. Vitreous amyloidosis is characterized by a close or loose phenotype with retinal adhesion. This project verifies the interaction of the two mutants by constructing the report plasmid. It is clear that there are other virulence genes in addition to the above two mutations, and whether the TTR gene is related to the cloudy phenotype of the ocular vitreous body. The relationship between the stability and expression of TTR protein and the incidence of HVA was explored by detecting retinol concentration and TTR protein expression in the eye tissue. This study will explore the genetic mechanism of the TTRGly83Arg mutation, which only leads to HVA, and enrich and supplement the understanding of the TTR expression and regulation in retinal cells.

家族性玻璃体淀粉样变性（HVA）是一种因转甲状腺素蛋白（TTR）基因突变致淀粉样混浊物异常沉积在玻璃体腔的遗传性疾病。能导致患者严重视力障碍，无有效的治疗方法。因此，进一步探索HVA的发病机制、寻找新的治疗靶点具有重要的意义。前期研究证实TTR Gly83Arg突变与HVA的发病相关；且该突变和TTR c.-743A>T突变均可导致血液中TTR蛋白表达下降；玻璃体淀粉样混浊物表现为与视网膜粘连紧密或疏松两种表型。该项目采用构建报告质粒的方式验证上述两突变的相互作用；全基因组外显子测序，明确除上述两突变以外是否还存在其它致病基因，TTR不同致病基因与眼部玻璃体混浊表型是否相关；通过检测眼球组织视黄醇浓度、TTR蛋白表达，探索TTR蛋白稳定性及表达量变化与HVA发病之间的关系。本研究将探索TTRGly83Arg突变仅导致HVA的遗传学学机制，并丰富和补充对视网膜细胞内TTR表达调控的认识。

目的：明确HVA新的致病基因，及不同致病基因与眼部玻璃体混浊表型的关系。探索TTR稳定性及表达变化与HVA发病之间的关系。.方法：针对TTR c.-743A>T（rs3794885）结合家系1发病特点进行基因型和表型关系分析。通过EMSA实验探索rs3794885的功能机制，色氨酸荧光及白藜芦醇结合荧光分析G83R突变对TTR结构稳定性的影响, PCR及WB检测TTR G83R突变模型小鼠TTR表达水平，色谱联合质谱测定眼球的视黄醇浓度，Pulldown明确G83R突变对TTR与RBP4结合能力的影响。.结果：（1）粘连紧密型的4名患者中有3人为c.-743A/T、1人为c.-743A/A；粘连疏松的2名患者中1人为c.-743A/T、另1人为c.-743A/A。TTR c.-743A>T突变与淀粉样物质粘连类型无严格的共分离现象。rs3794885所在片段不能与转录因子SP1结合。（2）全外显子测序发现家系1和2的一系列的可能致病基因。经分析明确了家系1和家系2的致病基因分别为TTR c.307G>C和c.311T>A。（3）三级结构稳定性由强到弱为TTR-wt、TTRG83R、TTRV30M，IC50盐酸胍浓度分别为5.3M、4.6M和3.0M；三级结构展开速度EC50时间分别为=9.6h、2.3h和2.1h。四级结构稳定性由强到弱为TTR-wt、TTRG83R、TTRV30M，IC50盐酸胍浓度分别为4.9M、4.1M和1.9M，四级结构解聚速度EC50时间分别为9.9h、1.9h和0.7h。与TTR-wt结合的RBP4明显较TTRG83R多，说明野生型TTR-RBP4结合率更高。（4）模型小鼠眼球中TTR表达水平约为野生型小鼠的60%（P<0.05）。模型小鼠眼球中视黄醇浓度150μg/L，野生型215μg/L，差异有统计学意义（P<0.05）。.结论：（1）TTR c.-743A>T突变与HVA患者淀粉样物质粘连类型无明确关系。rs3794885所在片段不能通过与SP1结合影响下游基因的表达。（2）TTR c.307G>C和TTR c.311T>A均为HVA的致病基因。（3）G83R突变导致TTR稳定性下降及TTR与RBP4结合力下降。（4）TTR G83R突变导致眼球组织TTR的表达水平下降，同时在一定程度上影响了视黄醇的转运使眼部视黄醇浓度降低。