Arc基因与我国汉族人群阿尔茨海默病的遗传相关性研究

发布时间：2018-01-02 16:31

本文关键词：Arc基因与我国汉族人群阿尔茨海默病的遗传相关性研究　出处：《安徽大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景阿尔茨海默病(Alzheimer's disease,AD)在我国俗称老年痴呆症,是一种最为常见的神经退行性疾病和痴呆类型,临床症状表现为记忆的缺失,以及不能形成新的记忆。研究发现,AD主要的病理特征是脑细胞外由Aβ异常聚集形成的老年斑和脑细胞内高度磷酸化tau蛋白聚集形成的神经纤维缠结。由于AD疾病进程伴随着大量的突触丢失,使得AD也被认为是一种"突触疾病"。Arc基因编码活性调节的细胞骨架蛋白,位于人的第八号染色体,属于即刻早期基因家族。Arc诱发突触的长时程增强效应,是突触可塑性、学习和记忆的关键调控因子。大量证据表明,Arc在记忆的巩固过程中发挥着重要作用。并且,前期研究发现,Arc蛋白还会和PSEN1相互作用,促进Aβ的生成。由于Aβ异常聚集形成的老年斑和记忆丢失是AD的典型病理特征,我们推测Arc基因的遗传变异可能与AD遗传易感相关。近期也有研究报道Ac基因的遗传变异在瑞典人群中与AD遗传易感相关。但是,到目前为止,没有关于Ac基因的遗传变异在我国汉族人群中与AD遗传易感相关的报道。方法本研究分两个阶段,一共收集了 1471例病例-对照样本,所有的样本均来自我国汉族人群,以探究Arc基因中的常见变异与AD的遗传易感相关性。同时,我们对99例具有高遗传力的AD样本(具有家族遗传史或发病年龄早于55岁)和52例认知正常的对照样本进行全外显子测序,并从中提取了Arc基因的全外显子测序结果进行分析,以探究在具有高遗传力的AD样本中是否存在Ac的稀有致病变异。对发现的变异,从GenBank中下载11种脊椎动物的Arc蛋白序列,应用软件包DNASTAR Lasergene7.1中的MegAlign软件比对蛋白序列,以探究在物种进化过程中Arc蛋白序列的保守性。使用RaptorX软件预测变异是否会潜在改变蛋白结构。通过挖掘分析基因表达数据库(Gene Expression Omnibus(GEO)和BRAINEAC),以探讨Arc蛋白在AD中的表达变化,以及AD相关变异位点与Ac基因表达的相关性,并通过双荧光报告实验对处于非编码区的调控位点进行功能初探。通过挖掘并分析ADNI数据库中180例AD患者、363例轻度认知功能障碍患者以及214例正常对照样本的全基因组关联分析(Genome-wide association study,GWAS)、临床数据以及生物指标数据,以探究风险等位基因与AD内表型的关联。结果对我们的样本研究发现,Arc基因3'UTR区变异位点rs10097505在我国汉族人群中与AD遗传易感显著相关(p=0.003)。通过GEO相关数据分析发现,在AD疾病进程中,颞叶皮层中的Arc表达水平显著降低;跨平台归一化分析其他表达数据集发现,在与AD发病最相关的脑区内嗅皮层中Arc的表达水平在AD人群中显著低于正常人群。而查询brain eQTL BRAINEAC数据库分析发现,SNP rs10097505风险等位基因型AA在海马中与显著上调的Arc mRNA的表达相关,这一发现得到了双荧光报告实验的验证。通过下载ADNI公开发表的数据,并进行整合分析发现,AD风险基因型rs10097505-AA较其他基因型携带者,在正常人群的脑脊液中与显著高的Aβ1-42的水平相关,同时与较高的Aβ水平的相关。并且,该风险基因型还在正常人群中与较低的MMSE(Mini-mental state examination,MMSE)认知评分相关。我们进一步通过外显子测序分析了Ar 基因外显子区的稀有变异,在两个无亲缘关系的AD患者中,发现了一个非同义变异c.20GA(p.T7I),该变异在认知正常的对照人群中未见报道。保守性分析显示,变异c.20GA改变了 Arc蛋白高度保守的第7位氨基酸,蛋白结构预测也显示该变异可能潜在影响蛋白结构。结论我们的研究发现,Arc基因中位于调控区的常见变异和位于编码区的稀有变异均与AD发病风险显著相关。细胞功能实验和数据挖掘分析显示,与AD遗传易感相关的Arc基因变异位点可能是通过改变Arc蛋白结构或者影响Arc在大脑中的表达,进而参与到AD发病机制中。接下来我们还需要独立样本人群的验证研究和系统的细胞和动物实验,来进一步揭示Arc参与AD发生发展的具体机制。
[Abstract]:The background of Alzheimer's disease (Alzheimer's disease, AD) in China, commonly known as Alzheimer's disease, is one of the most common neurodegenerative disease and dementia, clinical symptoms of memory loss, and unable to form new memories. The study found that the main pathological feature is AD tangles highly phosphorylated tau protein aggregation formation of brain cells by A beta abnormal aggregation formation of senile plaques and brain cells. Because of the AD disease process is accompanied by a large number of synaptic loss, the AD is also considered a "regulation of synaptic.Arc gene encoding disease activity of cytoskeletal proteins, located on human chromosome eighth, which belongs to the immediate early gene family.Arc evoked synaptic long-term potentiation, is a key regulator of synaptic plasticity, learning and memory. A lot of evidence that Arc plays an important role in the consolidation process, The preliminary study found that Arc protein can interact with PSEN1, promote the formation of A beta. Because A beta abnormal aggregation formation of senile plaques and memory loss is the typical pathological features of AD, we speculate that the genetic variation of Arc gene may be associated with AD genetic susceptibility. A recent study on genetic variation of Ac gene reported in Sweden people with AD genetic susceptibility. However, so far, no genetic variation of Ac gene in Chinese Han population and AD genetic susceptibility reports. Methods this study was divided into two stages, a total collection of 1471 cases of all the samples were the same, from China's Han the crowd, to explore the common AD genetic variation and Arc gene in susceptible correlation. At the same time, we have high heritability of 99 cases of AD samples (with family history or early onset age in 55 years) and 52 cases of normal cognitive on the same Whole exome sequencing, and extracted from all exons of Arc gene sequencing results were analyzed to explore whether the high heritability of AD samples are rare. The pathogenic mutation of Ac found mutation, Arc protein sequences downloaded 11 vertebrate species from GenBank, MegAlign software application software package comparison of protein sequences of DNASTAR in Lasergene7.1, to explore the Arc protein sequence in the evolution of species conservation. Using RaptorX software to predict the variation of whether potential changes in protein structure analysis. The expression of gene database by mining (Gene Expression Omnibus (GEO) and BRAINEAC), the expression of Arc protein in AD, and the correlation between AD mutation and Ac gene expression, and through the experiment of double fluorescence report function in non encoding region. Regulatory sites through mining and analysis in 180 cases of AD ADNI database Analysis of 363 cases of patients with mild cognitive impairment and 214 normal control samples of whole genome association (Genome-wide association study, GWAS), the clinical data and biological data, to explore related risk allele and AD phenotype. The results of study we found that Arc 3'UTR gene mutation in my rs10097505 in the Chinese Han population and AD genetic susceptibility significantly correlated (p=0.003). Through the GEO data analysis found that in the AD disease process, the expression level of the temporal cortex in Arc decreased significantly; the normalized cross platform analysis of other expression data sets found in the expression level and the incidence of AD in brain regions associated with most of the entorhinal cortex Arc in the AD population was significantly lower than that of normal people. Brain eQTL BRAINEAC database query and analysis found that the SNP rs10097505 risk allele AA was significantly up-regulated in the hippocampus of Arc and mRNA The expression, this finding was verified by experiments. A double fluorescence report published by downloading the ADNI data integration and analysis found that the risk of AD genotype rs10097505-AA than other genotype, in normal population in CSF was significantly higher and A beta 1-42 level, at the same time with higher A beta the level of correlation. And, with the lower MMSE in the normal population risk genotype (Mini-mental state examination, in MMSE). We further cognitive score by exon sequencing analysis of rare mutations in Ar gene exon region, in two unrelated AD patients, found a non synonymous mutation c.20GA (p.T7I), the variation reported in control group normal cognitive. Conservative analysis shows that the variation of c.20GA changed seventh amino acid Arc highly conserved protein, protein structure prediction also shows the change Different potential effect of protein structure. Conclusion our study found that located in the promoter region of Arc gene in common variants and is located in the encoding region of rare variants were significantly associated with the risk of AD. Cell function experiments and data mining analysis showed that the susceptible and AD genetic variation in Arc gene loci related probably by changing the structure of Arc protein or the effect of Arc expression in the brain, and then participate in the pathogenesis of AD. Cell and animal experiment study and then we need independent population system, to further reveal the specific mechanism of Arc involved in the occurrence and development of AD.

【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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