Development and Clinical Application of Molecular Diagnostic Tests

越来越多的基因与阿尔茨海默病-S病(AD)有关。最近，人类α2-巨球蛋白基因(A2M)的一种新的多态性被认为是AD的危险因素。这种多态是由于外显子24的A到G的转换，根据cDNA1000位的序列或976位的成熟蛋白。该点突变使α2-巨球蛋白的硫酯位点附近的Ile(ATC)变为Val(GTC)。由此产生的多态频率很高，在白人群体中，等位基因分布为60%到70%的A，而不是30%到35%的G。我们开发了一种实用的方法来检测这种多态。该方法基于半自动聚合酶链式反应-单链序列多态分析，比以往的方法，如聚合酶链式反应-限制性片段长度多态分析和DNA直接测序，具有更快和/或更简单的特点。用这三种方法对人外周血标本进行的平行分析表明，新方法的诊断结果与以前的方法一样可靠。因此，新的方法特别有利于大规模研究这种新的点突变与AD风险之间的关系。除了基因异常，一些感染性病原体，如单纯疱疹病毒1型(HSV-1)已被认为参与了AD的发生。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，从老年AD患者和对照组的颞叶和额叶皮质编码的大脑样本中，只有一小部分HSV-1 DNA阳性。此外，在AD和载脂蛋白E4患者的大脑中，HSV-1DNA并不比未受影响的患者更常见，这表明HSV-1在AD中不太可能起到作用。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?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.