| 题名 | 线粒体基因与阿尔茨海默病遗传关联分析 |
| 作者 | 毕蕊 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 姚永刚 |
| 关键词 | 阿尔茨海默病 mtDNA 单倍型类群 PARL 细胞色素c氧化酶 |
| 其他题名 | Association study of mitochondrial genes in Han Chinese with Alzheimer’s disease |
| 中文摘要 | 阿尔茨海默病 (Alzheimer's disease, AD; OMIM: 104300)，也被称作老年痴呆症，是发生在老年前期与老年期的一种原发性退行性脑病，主要表现为短期和长期记忆逐渐丧失，是最为常见的一种痴呆类型。在我国65岁以上的人群中有3.21%的人为AD患者，并且我国正进入老龄化社会，AD的发病率逐年快速上升将是不容忽视的问题，并会带来沉重的社会与家庭负担。 线粒体是细胞的能量工厂，其功能异常会对高耗能的神经系统产生影响，从而导致一系列神经退行性疾病的发生。在AD中线粒体的功能异常已经是被大量研究共同认可的事实，也被认为是导致AD的原因之一。然而，目前还较少有研究关注线粒体基因在AD遗传易感中的作用。本文从线粒体角度出发，分别从线粒体DNA (mitochondrial DNA, mtDNA)遗传背景以及核编码的线粒体基因两方面对线粒体基因与AD的易感性进行了研究，目的是想探索有哪些参与到AD致病通路的线粒体基因，这些基因是否也会在AD遗传易感方面有所贡献。 mtDNA遗传背景可以影响很多神经退行性疾病的发病风险，目前关于mtDNA遗传背景与AD的研究都集中于欧美人群，并且没有得到一致的研究结果。我们分析了在来自我国西南和东部地区的两个相互独立的汉族AD病例 (n=712)和正常对照 (n=905)群体的mtDNA遗传背景，发现单倍型类群B5在两个AD病例对照群体以及合并的我国汉族群体中都与AD发病风险显著相关 (P = 0.02; OR =1.74; 95% CI = 1.10-2.76)；系统地理学研究也发现我国各省AD的患病率和单倍型类群B5的频率分布有部分的重叠效应。对永生化细胞的线粒体功能检测发现，B5a类群的永生化细胞比起其他类群的细胞表现出显著的线粒体功能下降，说明单倍型类群B5和其亚类群可以影响线粒体功能。我们推测单倍型类群B5的界定位点m.8584G>A (MT-ATP6, p.A20T)是导致B5类群和AD相关的潜在原因。针对ATP6基因的异位表达实验证实了我们的假设，即稳定表达8584A等位基因的细胞株比起稳定表达8584G等位基因的细胞株，线粒体功能显著降低。我们的结果说明，B5类群的界定位点m.8584G>A可能通过影响线粒体功能而成为介导B5类群AD易感的潜在原因，这也为古老变异疾病易感性理论提供了更多的实验佐证。 基于帕金森病 (Parkinson’s disease, PD)和AD都是与线粒体相关的神经退行性疾病，且二者在发病机制上有一定的相似性，我们假设和PD相关的线粒体基因也有可能影响AD的遗传易感。因此我们选择了和PD相关的且为线粒体自噬、凋亡以及融合分裂等通路中的关键基因LRRK2、PINK1和PARL，在收集的我国汉族AD病例和正常对照样本中进行分析。结果发现LRRK2和PINK1基因中的遗传多态位点 (Single Nucleotide Polymorphism, SNP)与AD风险不相关，而PARL基因中有4个SNP位点与AD易感相关。其中位于PARL基因上游约1200 bp的位点rs7653061在多个AD病例对照群体中都与AD风险显著相关，且该位点不同基因型与内嗅皮层厚度以及大脑萎缩程度显著相关。eQTL分析发现，PARL基因中和AD遗传易感相关的位点rs3749446的风险型基因型TT和人多个脑区显著降低的PARL mRNA水平相关，而该风险基因型同时还和显著降低的认知水平和海马体积，以及显著升高的脑脊液β淀粉样蛋白 (β-amyloid, Aβ)水平相关。进一步的细胞水平实验发现，经Aβ1-42处理的U251细胞PARL基因的表达水平显著降低，而过表达PARL的U251细胞Aβ1-42生成显著减少。我们的结果提示，PARL和AD遗传易感相关，并且PARL的AD风险基因型与降低的PARL表达水平以及升高的Aβ水平显著相关，这一相关性也得到了细胞水平实验的验证。 鉴于大量研究均发现线粒体呼吸链复合物IV，即细胞色素c氧化酶可能参与到AD的致病通路中，我们在我国汉族AD病例和正常对照样本中对该酶的17个组成亚基以及装配因子进行分析。结果发现细胞色素c氧化酶装配因子SURF1和COX10，以及细胞色素c氧化酶亚基NDUFA4基因与我国汉族AD遗传风险相关。其中，NDUFA4基因还被发现在衰老和AD疾病进程中表达水平逐渐下降，提示该基因有可能在衰老和AD过程中发挥重要作用。 综上所述，我们在我国汉族人群中对线粒体相关基因与AD的遗传易感性进行了关联分析。发现mtDNA遗传背景B5，以及核编码的线粒体基因PARL、SURF1、COX10和NDUFA4与我国汉族AD发病风险相关。我们的研究为线粒体基因与AD的相关性提供了更多的遗传学证据。接下来扩大样本量的研究以及对易感基因和位点的功能验证将有望为AD的早期诊断和治疗提供更多的基础数据。 |
| 英文摘要 | Alzheimer’s disease (AD, OMIM 104300) is the most common kind of dementia, which mainly leads to severe memory loss in elderly people over 65 years old. Due to the important roles of mitochondria in cellular metabolism and energy production, the nervous system, with high energy demands, is especially vulnerable to mitochondrial defects. In fact, mitochondrial dysfunction is widely reported in neurodegenerative diseases including AD. In this study, we aimed to investigate whether mitochondrial genes confer genetic susceptibility to AD in Han Chinese. Mitochondrial DNA (mtDNA) haplogroups were identified to affect susceptibility of AD in European populations. However, there is still no related study in Chinese population. In this study, we investigated the association between matrilineal structures of Han Chinese populations and AD by a two-stage case-control study: 341 AD patients and 435 normal individuals from Southwest of China were analyzed for mtDNA sequence variations and were classified into respective haplogroups. 371 AD patients and 470 normal individuals from East China, as validation samples, were genotyped for the variants defining the risk haplogroup. Haplogroup B5 had a significantly higher frequency in AD patients (7.33%) than in controls (3.68%) from Southwest China, and we found a similar pattern of higher frequency of B5 in patients in the case-control sample from East China. In the combined population, association of haplogroup B5 with AD risk was strengthened (P=0.02; OR=1.74; 95% CI=1.10-2.76). In lymphoblastoid cell lines belonging to haplogroup B5a, we observed significantly increased reactive oxygen species (ROS) and decreased mitochondrial mass. Hela cells with stable expression of the MT-ATP6 gene with B5 defining variant m.8584G>A also showed a significantly decreased mitochondrial function. Taken together, our results indicated that haplogroup B5 conferred genetic susceptibility to AD in Han Chinese and this effect was most likely mediated by ancient variant m.8584G>A. The predisposing effect of B5 to AD is consistent with the ancestral-susceptibility model of complex diseases. Considering that AD and Parkinson’s disease (PD) are two typical neurodegenerative diseases with some overlapping pathologies, we further screened single nucleotide polymorphisms (SNPs) in the LRRK2, PINK1 and PARL gene to investigate whether these PD related mitochondrial genes could also pose a risk for AD. Results indicated that SNPs in the LRRK2 and PINK1 gene were not associated with genetic risk of AD, while SNPs in the PARL gene were significantly associated with AD risk. The positive association between SNP rs7653061 and AD was verified in several independent AD case-control cohorts. Further data mining of the ADNI database revealed that SNP rs7653061 was significantly related to altered entorhinal volume and brain atrophy. eQTL data of SNP rs3749446 in the PARL gene showed that risk genotype TT was significantly associated with reduced brain PARL mRNA level. Furthermore, rs3749446 was identified to be related to significantly increased β-amyloid (Aβ) in cerebrospinal fluid (CSF), and significantly decreased hippocampal volume and cognitive ability. The association between PARL and AD was further verified by functional experiments in U251 cells. mRNA level of PARL in U251 cells was significantly down regulated under Aβ1-42 treatment, while overexpression of PARL in U251 cells lead to significantly decreased Aβ1-42 production. Large amount of studies have observed severe perturbations in the mitochondrial electron transport chain in AD, especially for complex IV (also called cytochrome c oxidase (COX)). In this study, we screened 41 SNPs of 17 COX subunit genes and assembly factor genes in AD cases and controls from Han Chinese. SNPs in COX assembly factor genes SURF1 and COX10, and subunit gene NDUFA4 were identified to be associated with AD risk. Furthermore, the mRNA level of NDUFA4 significantly reduced du |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10176]  |
| 专题 | 昆明动物研究所_重大疾病机理的遗传学 |
| 推荐引用方式 GB/T 7714 | 毕蕊. 线粒体基因与阿尔茨海默病遗传关联分析[D]. 北京. 中国科学院研究生院. 2014. |
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