Development/ Application Of Molecular Diagnostic Tests

在过去的十年里，越来越多的基因与阿尔茨海默病(AD)有关。尽管有相反的证据，但最近的一些病例对照研究表明，组织蛋白酶D(cathepsin D，CATD)基因外显子2的C-T多态增加了散发性AD的风险。组织蛋白D编码基因位于11号染色体短臂末端，位于p15带。该基因转录部分长约11,000个碱基，由9个外显子组成，与人类胃蛋白酶原A基因相似。组织蛋白酶D是一种存在于细胞内体-溶酶体系统中的主要天冬氨酸蛋白酶。CATD基因外显子2的C-T多态十分常见，导致第224位氨基酸序列由Ala变为Val。T等位基因可能与蛋白表达增加(CAT D原分泌增加)和细胞内成熟改变有关。我们建立了检测CATD基因外显子2 C-T多态的新的、实用的方法。用新设计的引物和/或不同的限制性内切酶，对患者和对照组外周血DNA进行常规的聚合酶链式反应-限制性片段长度多态性分析，显著缩短了孵育时间，使孵化温度从60oC降至37oC。基于聚合酶链式反应-单链构象多态性(PCR-SSCP)检测，我们还建立了一种半自动的检测方法。结果表明，在优化的条件下，该方法与PCR-RFLP和DNA直接测序方法一样可靠，但操作简单、快速。此外，我们开发并验证了一种基于LightCycler仪器的全自动、实时超快聚合酶链式反应方法，用于检测CATD基因的C到T序列多态性。随着自动化程度和速度的提高，后两种方法将成为常规实验室和大规模筛查的良好替代品。关于感染因素在AD发病中的可能作用，我们的合作研究表明，AD患者脑内单纯疱疹病毒1型(HSV-1)DNA和载脂蛋白E4等位基因的发生率没有增加，这使得HSV-1参与AD的可能性不大。在其他合作研究中，我们继续研究载脂蛋白(A)亚型和等位基因在动脉粥样硬化性心脏病和系统性红斑狼疮患者中可能的致病作用。

An increasing number of genes have been linked to Alzheimer disease (AD) over the past decade. Although there is contrary evidence, some recent case-control studies suggested that a C to T polymorphism in exon 2 of the cathepsin D (CATD) gene increases the risk of sporadic AD. The gene encoding cathepsin D is located at the extremity of the short arm of chromosome 11, in the p15 band. The transcribed portion of the gene is about 11,000 bp and it is organized into 9 exons analogous with the human pepsinogen A gene. Cathepsin D is a major intracellular aspartyl protease present in the endosomal-lysosomal system. The C to T polymorphism in exon 2 of the CATD gene is common and results in an amino acid sequence change at residue 224 from Ala to Val. The T allele may be associated with increased protein expression (increased pro-cat D secretion) and altered intracellular maturation. We developed new and practicable methods for detection of the C to T polymorphism in exon 2 of the CATD gene. Using newly designed primers and/or a different restriction endonuclease, we markedly shortened the incubation time and reduced the incubation temperature from 60 oC to 37 oC in conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of DNA extracted from peripheral blood of patients and their controls. Based on PCR-single strand conformation polymorphism (PCR-SSCP) detection, we also developed a semi-automated method. We showed that, under optimized conditions, this method is as reliable as PCR-RFLP and direct DNA sequencing but is simpler and faster to perform. Further, we developed and validated a fully automated, real-time ultrafast PCR method based on the use of the LightCycler instrument for detection of the C to T sequence polymorphism in the CATD gene. With their increased level of automation and speed, the latter two methods would be favorable replacements for PCR-RFLP and direct DNA sequencing for both routine laboratory use and large-scale screening. Regarding the possible role of infectious agents in the development of AD, our collaborative study showed no increased incidence of Herpes simplex type 1 virus (HSV-1) DNA in the brains of patients with AD and apolipoprotein E4 allele, making the participation of HSV-1 in AD unlikely. In other collaborative studies, we continued studying the possible pathogenetic role of apolipoprotein(a) isoforms and alleles in patients with atherosclerotic heart disease and systemic lupus erythematosus.