Development And Clinical Application Of Diagnostic Tests

在过去的十年里，越来越多的基因多态与阿尔茨海默病有关。利用杂交探针进行实时聚合酶链式反应(PCR)和熔化曲线分析是常规诊断这些基因多态性的重要工具。此前，我们开发了一种LightCycler方法来研究组织蛋白酶D基因的多态与阿尔茨海默病之间的可能关系。组织蛋白酶D是一种存在于内体-溶酶体系统中的主要胞内蛋白水解酶。它的活性仅限于酸性pH，在疏水氨基酸附近有强烈的切割倾向。组织蛋白酶D基因外显子2(编码序列)的一个常见的多态(C到T)将224位残基的酶产物中的氨基酸序列从丙氨酸改变为缬氨酸。T等位基因可能与蛋白质表达增加(组织蛋白酶D原分泌增加)和细胞内成熟改变有关，已被认为是阿尔茨海默病的标志。在对500多名受试者的研究中，我们发现了一例组织蛋白酶D基因的变异模式-LightCycler熔化图谱，防止了等位基因歧视。因此，在有异常熔化曲线的受试者中，DNA测序或限制性片段长度多态性(RFLP)分析是必要的，以明确指定正确的组织蛋白酶D基因。 在一项合作研究中，我们研究了脑脊液β-淀粉样蛋白(1-42)和tau水平(两个？已建立的阿尔茨海默病标志物？)的稳定性。阿尔茨海默病患者载脂蛋白E基因分型的研究

An increasing number of gene polymorphisms have been linked to Alzheimer disease over the past decade. Real-time polymerase chain reaction (PCR) followed by melting curve analysis using hybridization probes is an important tool in routine diagnosis of these polymorphisms. Previously, we developed a LightCycler method for the study of a possible relationship between polymorphism of the cathepsin D gene and Alzheimer disease. Cathepsin D is a major intracellular protease present in the endosomal-lysosomal system. Its activity is limited to acidic pH with a strong preference for cleavage near hydrophobic amino acids. A common polymorphism (C to T) in exon 2 (a coding sequence) of the cathepsin D gene changes the amino acid sequence in the enzyme product at residue 224 from alanine to valine. The T allele may be associated with increased protein expression (increased pro-cathepsin D secretion) and altered intracellular maturation and has been suggested to be a marker for Alzheimer disease. During the study of more than 500 subjects, we identified a case with a variant pattern of the cathepsin D gene-LightCycler melting profile preventing allelic discrimination. As a consequence, in subjects with unusual melting curve profile DNA sequencing or restriction fragment-length polymorphism (RFLP) analysis is necessary to unequivocally assign the correct cathepsin D genotype. In a collaborative study, we studied the stability of cerebrospinal fluid beta-amyloid(1-42) and tau levels (two ?established Alzheimer markers?) by apolipoprotein E genotype in patients with Alzheimer disease.