【作者】 卢晓雄；

【导师】 张宝荣；

【作者基本信息】 浙江大学 ， 神经病学， 2012， 硕士

【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。更多还原

【Abstract】 Background:Alzheimer’s disease (AD) is a progressive degenerative disorder of the centr al nervous system with progressive loss of memory, impairment of cognitive fun ction, and changes in personality amony its clinical features. The pathology of A D is characterized by the aggregation and deposition of amyloid-β (AP) peptide r esulting in the formation of extracellular neuritic plaques and hyperphosphorylates tau protein resulting in the formation of intracellular neurofibrillary tangles. Mut ations in the amyloid-β precursor protein, presenilin-1(PS-1), and presenilin-2ge nes have been linked to autosomal dominant familial AD (FAD), with the most common mutations occurring in PS-1. However, the genetics of sporadic AD are less clear, with only the apolipoprotein E (ApoE)4allele considered to be a ke y risk factor in its etiology. In addition, some research has found individuals wit h sporadic AD who have PS-1mutations. The prevailing view of the effects of PS-1mutations in AD is that these mutations increase the production of amyloid-P42(AP42), triggering the pathogenic cascade, by causing gain of toxic function. On the other hand, more recent work has shown that many PS-1FAD mutations have no significant effect on the production of AP42. The precise mechanism, h owever, remains unknown. Additional evidence is needed to clarify the pathogeni c mechanism of PS-1mutations in AD.Objective:We analyzed the13exons and the splice junctions of PS-1for111cases of AD unselected for family history or age at onset to find new PS-1gene mutations.A novel heterozygous initiation codon mutation (from ATG to GTG) was found. mRNA expression levels of PS-1and APP gene and the PS-1proteins expression were detected by quantitative real-time PCR and western blot analysis in transfected cells to investigate the influence of the initiation codon mutation on the expression of PS-1.Methods:A total of266volunteers participated in our study, including111individuals with AD unselected for family history or age at onset, five individuals who were relatives of one of the111patients, and150healthy controls. Genomic DNA was extracted from peripheral blood leukocytes and collected from all participants using the phenol-chloroform extraction method. All PS1gene exons and the flanking intronic sequences were amplified by polymerase chain reaction (PCR). PCR products were direct sequenced by the Sangon Biotech Co. Ltd sequencing service.cDNAs encoding full-length wild-type PS-1and with a GTG as the initiation codon were subcloned into pEGFP-N1. HEK293T and N2a cells were transiently transfected with the empty vector (transfection control) or the confirmed vectors containing either wild-type or mutant PS-1cDNA using Attractene Transfection Reagent according to the manufacturer’s instructions. the GFP fusion proteins were detected by western blot analysis.Results:(1) We analyzed the sequences of the13exons and the splice junctions of PS-1for all111AD cases and found a heterozygous A to G mutation in the i nitiation codon in patient M766. The mutation was also detected in three unaffec ted relatives, but not in two other unaffected siblings,110cases or150healthy controls. The proband, a65-year-old male, had the first symptoms of mild memo ry impairment at the age of57years and had a gradual progressive cognitive di sorder that made him unable to recognize family members6years later. While t he proband’s93-year-old mother and63-year-old younger sister carrying the sam e mutation were both healthy. In addition, three previously reported polymorphis ms were found (rs1800839, rs116882898, and rs65932).(2) We analyzed mRNA expression levels of PS-1and APP gene in transf ected cells.The mRNA expression of mutant PS-1gene was significantly lower t han normal PS-1gene and the APP gene was not obviously altered.(3)The mutant PS-1with GTG as the initiation codon could be expressed in HEK293T, but not in N2aConclusion:(1) The unaffected carriers in our study may provide evidence that the mutation of PS-1does not cause AD and it might serve as a risk factor.Since no other mutations or polymorphisms were detected in our patiens, mutations in the coding regions and splice junctions of PS-1are likely to be rare in AD.(2) In our study, the mutation was also observed in three unaffected relatives as well as one individual with AD and all the carriers showed normal neuronal function including the individual’s mother and younger sister at the age above the onset age of the proband. It seemed that the mutation failed to segregate with the disease.(3) We have detected the expression of mutant cDNA carrying GTG as the start codon in HEK293T cells, but not in N2a cells. These data suggest that the GTG initiation codon is inefficient in neuron-like cells.(4) The results of mRNA expression levels of PS-1and APP may suggested that overexpression of PS-1do not affect the expression of APP.更多还原