背景： 阿尔兹海默病（Alzheimer´s Disease, AD）是与增龄相关、以渐进性认知功能障碍和记忆减退为主要症状、最常见的痴呆类型。作为老年期复杂中枢神经系统退行性变性疾病，AD具有多基因遗传背景，且发病机制复杂。胆固醇代谢稳态失调与认知功能障碍和AD有关，胆固醇调节相关的遗传变异也关联认知功能障碍和AD风险，然其机制依然不清。因此，在基因组和转录组水平开展AD病因学相关的系统研究将有助于探索AD的病因、生物标志物和致病机制，具有潜在的临床实际应用价值。
目的： 1、在中国AD人群中识别新的AD风险相关胆固醇调节基因遗传变异及验证候选AD风险相关基因变异并进行功能分析。2、在课题组前期研究基础上开展胆固醇调节基因APOC1体外分子机制研究，初步探索AD潜在的致病机制。3、探索功能相关AD转录组水平差异表达谱。
方法： 1、在854例AD患者、1059例AD源性轻度认知功能障碍（MCI-AD）患者和1254例认知正常老年对照共计3167例样本中，对12个胆固醇调节相关核受体基因和APOE共计13个基因进行靶向测序，筛选潜在变异并进行识别、验证和初步功能分析。2、利用分子微阵列质谱分型平台，在183例AD、255例MCI-AD患者和1544例对照共计1982例样本中，针对93个2015-2017年国际上报道但尚未在中国人群验证的、包括多个胆固醇代谢调节相关基因的遗传变异在内的、新的AD风险相关基因变异进行人群验证和致病性分析。3、对本组前期已证明关联AD风险的胆固醇调节基因APOC1，构建APCO1基因miRNA干扰重组慢病毒和APOC1基因过表达重组慢病毒分别感染MRC-5细胞，建立APOC1敲减和过表达的MRC-5细胞稳转系，600μmol/L双氧水处理后检测细胞增殖抑制、凋亡相关指标，如DNA复制活性、细胞凋亡小体、细胞质细胞色素C水平、活化的Caspase 3和Caspase 8表达量；建立APOC1敲减和过表达瞬时感染HepG2细胞模型，30mmol/L葡萄糖刺激后检测线粒体功能状态相关指标，如线粒体呼吸链酶复合物I（Complex I）活性、线粒体膜电位变化。4、对29例AD患者、12例MCI-AD患者和4例对照共计45例样本的外周血RNA进行链特性RNA-seq和生物信息学分析，初步分析AD特异血液转录组表达谱。
结果： 1、发现一个新的胆固醇代谢相关核受体基因低频变异，ESR1 rs9340803 A>G（MAF: 0.38%）独立或协同APOEε4同时增加AD和MCI-AD患病风险[OR(95%CI):3.32(1.80~4.05), p<0.001；OR(95%CI):4.69(1.39~5.89), p<0.001]，并与疾病表型相关联，初步功能分析显示该变异可降低ESR1基因转录调控活性。2、发现胆固醇转运相关载脂蛋白基因低频变异CLU rs117389184 C>T（MAF: 0.50%）同时增加中国AD和MCI-AD患病风险[OR(95%CI):3.20(1.21~4.59), p=0.013]，该变异可能会干扰转录因子结合，影响基因转录、表达。3、功能研究方面，成功构建了胆固醇调节基因APOC1敲减和过表达细胞模型，和对照组相比，APOC1过表达组细胞凋亡比例增高，增殖率降低，细胞质细胞色素C、活化的Caspase 8和Caspase 3表达升高，Complex I活性下降；APOC1敲减组细胞凋亡减少，增殖增多，细胞色素c表达降低，Complex I活性升高。4、相较于对照组，AD和MCI-AD患者存在差异表达基因622个（上调272个，下调350个）和差异表达的长非编码RNA（lncRNA） 889个（上调341个，下调548个），且与脂质代谢异常相关疾病及表型、神经退行性变性疾病等相关因素有关。
结论： 1、首次发现胆固醇调节基因变异同时影响AD和MCI-AD的患病风险:在基因组水平，在AD-MCI-AD-对照队列人群中识别和验证了两个与AD和MCI-AD风险共享的低频变异ESR1 rs9340803 A>G、CLU rs117389184 C>T，这两个变异可降低基因转录和表达，可能有病因学意义。2、在胆固醇调节基因细胞模型的体外研究中，APOC1表达增多损害线粒体功能，促进细胞凋亡，初步揭示了AD潜在的致病机制可能与APOC1参与胆固醇水平及分布稳态调节，影响线粒体功能和细胞凋亡有关。3、在AD-MCI-AD-对照人群血液转录组研究中，检测到AD和MCI-AD具有特异性差异表达谱，得到了分子功能和可能的发病机制的线索。以上研究结果可为进一步开展具有潜在的临床实际应用价值的研究提供依据。

background: alzheimer´s disease (ad) is the most common type of dementia among the oldest, which is featured with aging-related progressive cognitive impairment and memory loss. as a complex neurodegenerative disease with polygenic background, the pathogenetic mechanisms of ad remains largely elusive. cholesterol metabolism homeostasis has been demonstrated to participate in ad etiology and variants of cholesterol metabolism regulating genes have been reported to modify ad risk, in which mechanisms involved warrant intensive exploration. systemic and extensive studies at genomic and tranomic level separately help to explore on ad pathology as well as biomarkers, which would have potential clinical application.
ive: 1) to screen and validate new genetic variations of cholesterol homeostasis-regulating genes, and to validate, in our ad samples, candidate ad risk-related genetic variants reported in western countries while has not been reported in chinese ad patients. 2) to establish in vitro cholesterol-regulating gene models, with which to study on the potential of cholesterol metabolism homeostasis-related genes on promoting cholesterol metabolism homeostasis and involved mechanisms, thus to explore preliminarily on the pathogenetic mechanisms of ad. 3) to analyze ad-specific differential expression profile.
methods: 1) we performed the targeted-sequencing of 12 nuclear receptor genes plus apoe, which were involved in cholesterol metabolism homeostasis modulation, in 854 ad patients,1059 mild cognitive impairment due to ad (mci-ad) patients and 1254 cognitively normal elderly controls to screen new ad susceptibility genetic variants of nuclear receptors encoding genes, and to explore preliminarily on the latent pathogenetic mechanism. 2) we genotyped and analyzed subsequently 93 ad risk-related genetic variants, which had been reported in western countries while not been reported in chinese ad patients between 2015-2017 period, in 183 ad patients,255 mci-ad patients and 1544 controls.3) we conducted apoc1 gene overexpression and knock-down by use of rnai, established mrc-5 cell models with decreased apoc1 expression and apoc1 overexpression, respectively, and tested classic cell proliferation and apoptosis related indicators like apoptosis body, cytochrome c, cleaved caspase 3 and caspase 8, after treatment of h2o2. we also established hepg2 cell models with apoc1 expression derceased and overexpressed, respectively, and tested mitochondrial function related indicators like complex i and mitochondrial transmembrane potential, after high glucose level stimulation. 4)we conducted strand-specific rna-seq of blood rna samples belonging to 29 ad patients,12 mci-ad patients and 4 controls from guangxi district, together with bioinformatic analyses, to analyze preliminarily ad specific blood tranome expression profile.
results: 1) we discovered a new low-frequency variant, esr1 rs9340803 at 6q25.1 (maf:0.38%), which´s both ad and mci-ad risk related [or (95%ci): 3.20 (1.84~4.22), p<0.001; or (95%ci): 3.08 (1.75~3.89), p<0.001]. this variant also correlated with disease phenotypes (p<0.05). preliminary functional study on it revealed decreased the tranion regulating activity of esr1 in vitro. 2) we discovered, for the first time, that clu rs117389184, a low-frequency snp (maf:0.5%), would increase the risk of chinese ad as well as mci-ad patients and associated with cognitive impairment [or (95%ci): 3.10 (1.14~3.30), p=0.02].this variant´s predicted to interfere with the binding of tranion factor and thus affect clu gene tranion.3) we established cell models with apoc1 overexpression and knock-down. preliminary results indicated that abnormal expression of apoc1 would effect on mitochondrial function and cell apoptosis. 4)we also discovered, for the first time, 622 differentially expressed genes (272 upregulated, 350 downregulated) and 889 differentially expressed long non-coding rnas (lncrnas; 341 upregulated, 548 downregulated) in ad and mci-ad patients from guangxi district by comparing with those of controls, within which immune response and multiple factors related to senile diseases were involved.
conclusions: 1) cholesterol homeostasis-regulating genes´variants would impact on both ad and mci-ad disease occurrence risk. at the gene level, we discovered, for the first time, that esr1 rs9340803 a>g and clu rs117389184 c>t could increase ad and mci-ad risk. these two variants would lead to decreased gene tranion and expression, separately. 2) cholesterol homeostasis-regulating gene apoc1 would have effect on cell apoptosis and mitochondrial dysfunction, which would result from disordered cholesterol metabolism. results of our study would offer evidence for explorations targeting potential clinical application. 3) at the tranome level, we also discovered, for the first time, that ad and mci-ad patients from guangxi district featured differential expression profile of blood. multiple factors including abnormal lipids metabolism related disease/phenotypes and neurodegenerative diseases related risk factors.