Development and Clinical Application of Molecular Diagnostic Tests

有许多基因与阿尔茨海默病（AD）有关。由于载脂蛋白E（apo E）基因的E4等位基因的存在被认为会增加AD的风险，因此apo E基因分型现在通常用于分子诊断实验室的阿尔茨海默病风险评估。尽管已经报道了多种方法，但apo E基因分型通常涉及聚合酶链反应（PCR）扩增的基因组DNA的限制性内切酶消化。这是一种相对简单的技术，但模式的解释通常是主观的，并且由于不完全的DNA消化、凝胶的可变DNA负载和检测技术的低灵敏度而变得复杂。用SYBR绿色染色代替溴化乙锭染色，提高了电泳条带检测的灵敏度。我们开发了一种定量的方法与图像分析，使带检测更客观。相对于最大的限制性片段定量各种限制性条带的密度，并建立决策水平以区分真实片段和部分消化的片段。最近，人类α 2-巨球蛋白基因的多态性也被证实为AD的危险因素。我们建立了一种新的、实用的检测该基因缺失多态性的方法。这种新方法是基于半自动PCR-单链多态性方法，我们表明，它是可靠的直接测序。在其他研究中，我们继续研究载脂蛋白（a）亚型和等位基因在心脏病患者和系统性红斑狼疮患者中的致病作用。

There are a number of genes that have been linked to Alzheimers disease (AD). Since presence of the E4 allele of the apolipoprotein E (apo E) gene is thought to confer increased risk of AD, apo E genotyping is now routinely used for Alzheimers risk assessment in molecular diagnostic laboratories. Although a variety of methods have been reported, apo E genotyping usually involves restriction endonu- clease digestion of polymerase chain reaction (PCR)-amplified genomic DNA. This is a relatively simple technique, but interpretation of the patterns often is subjective and is complicated by incomplete DNA digestion, variable DNA load of the gels, and low sensitivity of detection techniques. We improved standardization of DNA load of the gels and increased the sensitivity of band detection by using SYBR Green instead of ethidium bromide staining. We made band detection more objective by developing a quantitative approach with image analysis. The density of various restriction bands was quantified relative to the largest restriction fragment and decision levels were established to distinguish true from partially digested fragments. Recently, a polymorphism of the human alpha2-macroglobulin gene was also iden-tified as a risk factor for AD. We developed a new, practicable method for the detection of a deletion polymorphism of this gene. This new method is based on a semi-automated PCR-single stranded polymorphism approach and we showed that it is as reliable as direct sequencing. In other studies, we continue studying the pathogenetic role of apolipoprotein(a) isoforms and alleles in patients with heart disease and in those with sytemic lupus erythematosus.