Development And Clinical Application Of Diagnostic Tests

在过去的十年中，越来越多的基因多态性与阿尔茨海默病有关。实时聚合酶链反应（PCR）和杂交探针熔融曲线分析是常规诊断这些多态性的重要工具。此前，我们开发了一种LightCycler方法来研究组织蛋白酶D基因多态性与阿尔茨海默病之间的可能关系。组织蛋白酶D是存在于内溶酶体系统中的一种主要的细胞内蛋白酶。它的活性仅限于酸性pH值，强烈倾向于在疏水氨基酸附近切割。组织蛋白酶D基因外显子2（编码序列）的常见多态性（C到T）使酶产物中残基224处的氨基酸序列从丙氨酸变为缬氨酸。T等位基因可能与蛋白表达增加（前组织蛋白酶D分泌增加）和细胞内成熟改变有关，并被认为是阿尔茨海默病的一个标志。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- lightcycler熔化谱的变异模式，以防止等位基因歧视。因此，在具有异常融化曲线的受试者中，有必要进行DNA测序或限制性片段长度多态性（RFLP）分析，以明确指定正确的组织蛋白酶D基因型。

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.