Development and Clinical Application of Molecular Diagnostic Tests

越来越多的基因与老年痴呆症有关？阿尔茨海默病（AD）。最近，一种新的人类α 2巨球蛋白基因（A2M）多态性被认为是阿尔茨海默病的危险因素。这种多态性是由于外显子24的A到G的过渡，在cDNA序列的第1000位或在成熟蛋白的第976位。这种点突变使α - 2巨球蛋白硫酯位点附近的Ile （ATC）变为Val （GTC）。由此产生的多态性频率很高，在白人群体中，等位基因分布为60 - 70%的A和30 - 35%的G。我们开发了一种可行的方法来检测这种多态性。新方法基于半自动聚合酶链反应(PCR)-单链序列多态性（SSCP）分析，比PCR-限制性片段长度多态性（RFLP）分析和直接DNA测序等早期方法更快和/或更简单。用这三种方法对人外周血标本的平行分析表明，新方法的诊断可靠性与早期方法一样高。因此，这种新方法对于大规模研究这种新的点突变与AD风险之间的关系特别有利。除基因异常外，许多感染因子如单纯疱疹病毒1型（HSV-1）被认为参与了AD的发展。我们重新研究了这种病毒在AD病因学中的可能作用。我们发现，老年AD患者和对照组的颞叶和额叶皮层编码脑样本中只有一小部分呈HSV-1 DNA阳性。此外，单纯疱疹病毒-1 DNA在AD和载脂蛋白E4患者的大脑中并不比未受影响的患者更常见，这表明单纯疱疹病毒-1在AD中的作用不太可能。在其他合作研究中，我们继续研究载脂蛋白(a)异构体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发病作用。

An increasing number of genes have been linked to Alzheimer?s disease (AD). Recently, a new polymorphism of the human alpha2-macroglobulin gene (A2M) was proposed to be a risk factor for AD. This polymorphism is due to an A to G transition in exon 24, at position 1000 based on the cDNA sequence or at position 976 based on the mature protein. This point mutation changes Ile (ATC) to Val (GTC) near the thiolester site of alpha2-macroglobulin. The resultant polymorphism is of high frequency with an allele distribution of 60 to 70 percent A vs. 30 to 35 percent G in white populations. We developed a practicable method for detecting this polymorphism. The new method is based on semi-automated polymerase chain reaction (PCR)-single strand sequence polymorphism (SSCP) analysis and is considerably faster and/or simpler than earlier approaches such as PCR-restriction fragment length polymorphism (RFLP) analysis and direct DNA sequencing. Parallel analysis of human peripheral blood specimens with the three methods showed that the new method is as reliable diagnostically as are the earlier methods. The new method is thus particularly advantageous for large-scale studies of the relationship between this novel point mutation and AD risk.In addition to gene abnormalities, a number of infectious agents such as herpes simplex type 1 virus (HSV-1) have been suggested to participate in the development of AD. We re-investigated the possible role of this virus in the etiology of AD. We found that only a small percentage of coded brain samples from the temporal and frontal cortex of elderly AD patients and controls were positive for HSV-1 DNA. Furthermore, HSV-1 DNA was not more common in the brains of patients with AD and apolipoprotein E4 than in unaffected patients, indicating an unlikely role of HSV-1 in AD.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.