阿尔茨海默病患者presenilin-1基因突变筛查及其起始密码子突变后表达的初步研究

发布时间：2018-04-03 14:14

  本文选题：阿尔茨海默病　切入点：presenilin-1基因　出处：《浙江大学》2012年硕士论文

【摘要】：研究背景： 阿尔茨海默病(Alzheimer's disease, AD)是一种进行性不可逆神经系统变性疾病,是老年痴呆最常见的病因。至今AD的确切病因和发病机制不明,目前已知载脂蛋白Eε4等位基因与迟发性AD相关,早发性家族性AD主要由β-淀粉样前体蛋白(P-amyloid precursor protein, βAPP)、早老素-1(presenilin-1, PS-1)及早老素-2(presenilin-2, PS-2)基因突变引起。目前对于PS-1基因突变如何致病还不清楚,之前的研究认为突变的PS-1通过“获得功能”(gain of function)影响β-淀粉样多肽1-42(Amyloid β1-42,Aβ42)而发病,即淀粉样蛋白级联假说(Amyloid Cascade Hypothesis),该假说认为异常的β淀粉样蛋白(amyloid-P peptides, Aβ)是触发病理级联反应并最终导致AD的第一步。Ap是由正常存在于细胞膜上的APP经p-和γ-分泌酶剪切后产生一系列长度在39～43个氨基酸的短肽,其中,Aβ42最容易聚集纤维化,是主要的致病蛋白。早老素(presenilin, PS)蛋白是γ-分泌酶复合物的活性亚基,如发生突变,可影响APP剪切而导致异常Aβ的产生和聚集。近期的研究发现,某些PS-1基因突变不通过Aβ42致病。因此,需要更多的研究参与阐述PS-1基因突变与AD的关系。 目的： 通过筛查突变频率最高的PS-1基因突变来观察111例来自浙江大学医学院附属第二医院神经内科住院或门诊被诊断为AD的患者并且未经家族史和发病年龄筛选的情况下PS-1基因的突变情况。筛查发现1例患者PS-1基因3号外显子起始密码子发生了杂合突变ATG→GTG,构建突变型质粒转染细胞观察PS-1起始密码子突变后PS-1和APP的表达变化情况。 方法： 研究对象共266例包括未经过家族史和发病年龄筛选的111例来自浙江大学医学院附属第二医院神经内科住院或门诊被诊断为AD的患者,5例M766患者亲属,150例正常对照。常规酚/氯仿提取法提取外周血DNA,对PS-1基因13个外显子及侧翼内含子DNA扩增,聚合酶链反应(polymerase chain reaction, PCR)产物纯化后直接测序分析。利用体外定点突变技术突变野生型PS-1cDNA并构建野生型和突变型PS-1增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)融合基因质粒,通过实时荧光定量PCR检测PS-1和APP基因在人胚胎肾(HEK)293细胞、小鼠成神经瘤细胞N2a中的mRNA表达情况,并通过Western blot技术观察野生型和突变型PS-1蛋白在两个细胞系中的表达变化情况。 结果： (1)发现患者M766的PS-1基因3号外显子起始密码子发生了杂合突变ATG→GTG。该患者的发病年龄为57岁。其93岁母亲、63岁的大妹妹和患者的小儿子也检测到同样的突变,但均未发病。同时,发现3个已报道的单核苷酸多态(Single Nucleotide Polymorphisms, SNP)(rs1800839,rs116882898,rs165932)。 (2)实时荧光定量PCR检测发现PS-1基因mRNA在HEK293细胞和N2a细胞中野生型均显著高于突变型,APP基因mRNA在两细胞系中未见明显差异。 (3)Western blot结果显示野生型和突变型PS-1在HEK293细胞中均表达,且突变型表达量较野生型少。而N2a细胞仅表达野生型PS-1。 结论： (1)对未经过家族史和发病年龄筛选的111例来自浙江大学医学院附属第二医院神经内科住院或门诊被诊断为AD的患者行PS-1基因突变筛查未发现新的或已经报道的致病突变,提示PS-1基因突变导致的AD占总AD患者比例较低。 (2)根据M766患者家系图显示,未见PS-1基因起始密码子突变和疾病AD共分离现象,推测其可能不是致病突变,而是AD的一个危险因素,也可能是一个SNP。 (3)实时荧光定量PCR检测发现PS-1基因mRNA在HEK293细胞和N2a细胞中野生型均显著高于突变型,Western blot技术检测PS-1蛋白表达显示野生型和突变型PS-1在HEK293细胞中均表达,且突变型表达量较野生型少。而N2a细胞仅表达野生型PS-1,提示PS-1基因非ATG起始密码子GTG在HEK293细胞中表达效率降低,而在神经样细胞N2a中可能引起PS-1蛋白不表达。 (4)实时荧光定量PCR检测发现APP基因mRNA在两细胞系中未见明显差异,提示PS-1基因起始密码子突变对APP影响不大。
[Abstract]:Research background:
Alzheimer's disease (Alzheimer's disease AD) is a kind of irreversible neurodegenerative disease is the most common cause of senile dementia. The exact etiology and pathogenesis of AD is unknown, currently known apolipoprotein E 4 allele associated with late-onset AD, early-onset familial AD is mainly composed of beta - amyloid precursor protein (P-amyloid precursor protein, P APP), presenilin -1 (presenilin-1, PS-1) -2 early old (presenilin-2, PS-2) caused by gene mutation. The mutation of PS-1 gene to pathogenesis is unclear, the research before that mutant PS-1 by "function" (gain of function) effects of beta amyloid polypeptide 1-42 (Amyloid beta 1-42, beta 42 A) and the incidence, namely the amyloid cascade hypothesis (Amyloid Cascade Hypothesis), the hypothesis that amyloid beta protein abnormalities (amyloid-P peptides, A beta) is the pathological cascade trigger counter The first step should be.Ap and eventually AD is normally present in the cell membrane of APP by p- and gamma secretase produces a series of length 39~43 amino acid peptide, the A beta 42 most likely to gather the fibrosis is the main pathogenic protein. Presenilin (presenilin, PS) protein is the active subunit of the gamma secretase complex, such as mutation of APP can affect the shear caused by abnormal aggregation and A beta. A recent study showed that some mutations in the PS-1 gene by A beta 42 pathogenesis. Therefore, more research is needed in this relationship between PS-1 gene mutation and AD.
Objective:
By screening the mutation of PS-1 gene in the highest frequency of mutation was observed in 111 patients from the Second Affiliated Hospital of Zhejiang University Department of Neurology inpatient or outpatient PS-1 gene was diagnosed with AD and without family history and age of onset of the absence of screening the mutation screening. 1 cases were found in exon 3 of PS-1 gene start codon occurrence the heterozygous mutation of ATG, GTG, construction of mutant plasmid transfected cells to observe the expression changes of PS-1 and APP mutation after PS-1 initiation codon.
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