| 其他摘要 | 背景阿尔茨海默病（Alzheimer’s Disease, AD），又称老年痴呆症，约占所有痴呆类型疾病的60%~70%。AD是一种进行性发展的神经退行性疾病，最常见的临床症状为短期和长期记忆缺失，患病风险随年龄增长显著增加。AD最主要的病理特征为细胞外Aβ异常沉积形成的老年斑和细胞内由高度磷酸化的Tau蛋白形成的神经纤维缠结，很多AD相关的致病研究都是围绕这两个病理特征展开。目前AD的发病原因尚不清楚，但80%的AD都受到遗传因素的影响，多个基因被认为与AD的发病风险相关。转甲状腺素蛋白（Transthyretin, TTR）是一种主要在肝脏和脉络丛中合成的分泌型蛋白，用于转运甲状腺素和视黄醇。TTR参与到多种与淀粉样蛋白相关的疾病中，也是经过大量前期研究鉴定的家族性淀粉样多发性神经病（Familial Amyloid Polyneuropathy, FAP）和家族性淀粉心肌病（Familial Amyloidotic Cardiomyopathy, FAC）等疾病的重要致病基因。由于AD也是由β淀粉样蛋白病变导致的疾病，因此TTR很有可能在AD致病通路中发挥重要作用。虽然前期研究已经发现TTR蛋白可能参与到Aβ结合与清除的通路中，但关于TTR与AD的遗传相关性，目前还存在争论。本研究中，我们在我国汉族AD病例和对照群体中对TTR基因外显子区进行了检测，目的在于探索TTR基因变异是否和AD有潜在相关性。方法我们收集了源于我国汉族人群的529份AD患者和334份正常人的样本，并对这些患者进行TTR基因的全外显子和部分启动子测序，检测AD患者中是否存在影响TTR表达或功能的变异，并对这些变异进行保守性分析和软件功能预测。同时，为了探索一些稀有变异的功能，我们构建了不同的表达载体，转染细胞，调控区的稀有变异用双荧光报告系统检测相对荧光活性变化，CDS区域的变异用Western Blot检测AD通路相关蛋白的表达变化。结果在863份样本的TTR测序结果中，我们共发现了25例变异，其中两组中共有的常见变异位点（MAF>0.01）有9例。χ2检验显示这9例变异的等位基因频率在两组间并无显著性差异。对稀有变异的分析显示， AD患者中稀有TTR变异的携带者多于对照群体。此外我们进一步对稀有变异进行功能预测和细胞水平功能验证，发现：① 位于外显子剪接区的c.200+4A>G可能影响TTR的剪接，导致TTR蛋白表达的下调。② 非同义变异p.A111V和p.V50M可能会影响TTR蛋白的结构，进而影响TTR的功能，而过表达突变型TTR基因的细胞，细胞上清Aβ水平相对于过表达野生型TTR基因的细胞有所上调，而过表达TTR基因的细胞，tau蛋白磷酸化水平也显著上调。这些AD通路中的重要蛋白的改变说明TTR基因在AD中有潜在的重要作用。③ 对启动子区和UTR区的变异进行检测发现，在AD患者中发现的启动子区变异c.-319C>A可能会影响转录因子的结合位点，双荧光报告实验发现携带c.-319C>A的启动子活性呈现显著的下调趋势，说明c.-319C>A变异可能影响TTR的表达水平，而3’UTR突变体c.*30C>T的相对荧光活性也显著下调，说明该变异可能影响TTR的表达后调控从而影响TTR的表达，这两例变异可能最终都通过影响TTR表达水平而与AD发病相关。结论通过对TTR基因进行外显子测序，我们在AD患者中发现一些和AD潜在相关的稀有变异，并且发现AD患者携带TTR基因上的稀有变异的频率比普通人群的要高，而且从这些变异的分布和我们的功能试验显示，AD患者携带的TTR稀有变异更有可能会影响TTR的功能和表达水平，从而更进一步影响到AD通路。尽管目前的数据还不足以揭示TTR蛋白在AD中发挥作用的具体机制，但我们的结果为前期TTR与AD相关的功能研究提供了新的遗传学证据。关于TTR在AD通路中所扮演的角色，需要进一步的研究来挖掘。; BackgroundAlzheimer’s Disease, also known as Alzheimer Disease, accounts for 60% to 70% of all cases of dementia. AD is a chronic neurodegenerative disease that almost occurs in elder people with the common symptom of short-term and long-term memory loss and its risk increases significantly with age. The neuropathological characteristics of AD patients are the extracellular plaques of β-amyloid (Aβ) and intracellular hyperphosphorylated tau protein, and centered on these two hallmarks many studies about AD have developed. Until today, the cause of AD is not clearly elucidated, but recently studies showed that it has a high genetic heritability. Transthyretin (TTR)was reported to be synthesized mainly in liver and choroids plexus, and served as a transporter of thyroid and retinol in blood stream and Cerebrospinal Fluid (CSF). The TTR involves in many amyloid diseases, and was comfirmed as an important susceptibility gene for Familial Amyloid Polyneuropathy (FAP) and familial amyloidotic cardiomyopathy ( FAC). Similarly, AD was considered to be caused by amyloid peptide Aβ aggregates. Therefore, TTR probably play an important role in AD pathology. Although previous studies have identified that TTR might participate in the pathway of binding and clearing Aβ, the genetic correlation between TTR and AD has not been proven yet. In our study, we screen the whole exons of the TTR gene in Han Chinese AD patients and normal controls, aiming to explore the potential relationship between TTR variants and AD.Methods529 AD patients and 334 normal controls from Han Chinese population were recruited in our study. All exons of the TTR gene and nearby region were directly sequenced to find out whether there exists variants which may influence the expression and function of TTR protein. Then, we performed an evolutionary conservation anlysis and functional predictions for these variants by online softwares. We constructed a series of expression vectors and studied their functions at the cellular level. Luciferase report assay was used to detect the function of variants in regulatory regions. Western Blot was used to investigated the change of AD pathway related proteins caused by the non-synonymous mutations in TTR gene.ResultsA total of 25 variants were identified in the TTR gene in 863 individuals. Genotype analysis showed no significant difference between the allele frequencies of 9 common SNPs (MAF>0.01) in the two groups. AD patients had a higher rare variants frequency than the controls. Further study, functional prediction and cellular experiments were conducted to reveal the roles of these rare mutations, we found that: ①Mutation c.200+4A>G might influence the constitutive splicing of TTR mRNA, and decrease the production of the TTR protein. ②Non-synonymous mutations p.A111V and p.V50M might effect the structure of the TTR protein and disturb its function. Comparing to cells which overexpressed wild type TTR gene, those overexpressed TTR mutants have up-regulated Aβ level in supernatant. A up-regulation of phosphorylation in Tau also appeared in all TTR-overexpressing cells. These results indicate that TTR play an important role in AD. ③Prediction of variants in the regulatory regions of the TTR gene showed that c.-319C>A might affect the binding site of some transcription factors. Luciferase reporter assay demonstrated that c.-319C>A may reduce the promoter activity. Mutant allele c.*30C>T cause a reduction of relative luciferase activity relative to the wild-type allele, indicating its negative effect in post-transcriptional regulation. Both mutations might be related to AD by reducing TTR expression.ConclusionBy sequencing the TTR gene, we identified some potetially AD-related rare variants and also discovered that AD patients have a higher frequency of rare mutations than the normal people. Combined their distribution pattern in the TTR gene and functional study, our analysis showed that rare variants in AD patients are more likely to impair the expression and function of TTR, thereby going one step further to affect AD pathway. Despite the present data are not enough to uncover the specific functional role of TTR in AD, our findings provided new genetic evidence to confirm the potential relationship between of TTR and AD. Further study is still needed to excavate the role TTR in AD pathogenesis. |
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