Development And Clinical Application Of Molecular Diagno

基因多态性的研究越来越多地用于诊断人类疾病，评估人类疾病的风险（例如，发现动脉粥样硬化易感基因），监测治疗干预（例如，检测癌症中最小残留疾病或移植后的嵌合），以及预测药物反应（药物基因组学）。最近，全基因组扩增技术已经可用，通过这种技术可以将微量的DNA扩增到适合基因测试和分析的数量（例如，基因分型和DNA测序）。我们开始研究这样一种商业技术（REPLI-g, Qiagen）的分析性能和诊断可靠性，该技术声称适用于纯化的基因组DNA、新鲜或干燥的血液、灰白色被毛和其他细胞来源（例如，口腔或组织培养细胞）。这种等温（即非热循环）技术是基于所谓的多次位移放大。多重位移扩增的关键成分是一种独特的DNA聚合酶（噬菌体29），它能够在不与基因组模板分离的情况下激活非常大（高达100千碱基）的DNA片段。我们的初步结果表明，这种全基因组扩增技术提供了高分子量DNA的均匀产量，适用于遗传研究。在另一项工作中，我们回顾了目前可用的最先进的核酸诊断检测方法和未来的方向。在一项合作研究中，我们研究了阿尔茨海默病患者的脑脊液β -淀粉样蛋白（1-42）和tau水平（两种已确定的阿尔茨海默病标志物）和载脂蛋白E基因型。脑脊液中β -淀粉样蛋白（1-42）和tau蛋白的含量在阿尔茨海默病患者和老年正常对照组之间存在差异。时间和载脂蛋白E基因型对这些生物标志物的影响仍在继续阐明。我们评估了20例轻中度阿尔茨海默病患者的脑脊液β -淀粉样蛋白（1-42）和tau蛋白，其中11例载脂蛋白e4阳性，9例载脂蛋白e4阴性，平均时间3.8年（范围1-11.1年）。在测量期间，与载脂蛋白e4阴性患者相比，载脂蛋白e4阳性患者的脑脊液β -淀粉样蛋白（1-42）水平较低，并且随着时间的推移水平下降。随着时间的推移，Tau水平是稳定的，没有显示出载脂蛋白E等位基因的影响。尽管这些结果是基于有限的临床样本，但脑脊液β -淀粉样蛋白（1-42）的进一步下降（即更异常）与脑脊液tau蛋白的稳定性在近四年的平均时间内的结合表明，随着时间的推移，β -淀粉样蛋白（1-42）和tau蛋白保持其作为诊断生物标志物的潜在用途。如果将脑脊液β -淀粉样蛋白（1-42）和tau蛋白作为治疗反应的衡量标准，则应考虑这些发现。

Study of gene polymorphisms is increasingly used for diagnosing human diseases, assessing the risk of human diseases (e.g., finding atherosclerosis susceptibility genes), monitoring therapeutic interventions (e.g., detecting minimal residual disease in cancer or chimerism after transplantation), and predicting drug response (pharmacogenomics). Recently, whole genome amplification techniques through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis (e.g., genotyping and DNA sequencing) have become available. We began studying the analytical performance and diagnostic reliability of one such commercial technique (REPLI-g, Qiagen) that is claimed to be applicable to purified genomic DNA, fresh or dried blood, buffy coat, and other sources of cells (e.g., buccal or tissue culture cells). This isothermal (i.e., non-thermal cycling) technique is based on so-called multiple displacement amplification. The key component of multiple displacement amplification is a uniquely processive DNA polymerase (phage 29) capable of activating very large (up to 100 kilobase) DNA segments without dissociating from the genomic template. Our initial results indicate that this whole genome amplification technique provides a uniform yield of high-molecular-weight DNA that is suitable for genetic studies. In another work, we reviewed currently available state-of-the art methods and future directions for nucleic acid-based diagnostic testing. In a collaborative study, we studied cerebrospinal fluid beta-amyloid(1-42) and tau levels (two established Alzheimer markers) and apolipoprotein E genotypes in patients with Alzheimer disease. Cerebrospinal fluid measures of beta-amyloid(1-42 )and tau differ between patients with Alzheimer disease and elderly normal controls. The effect of time and apolipoprotein E genotype on these biomarkers continues to be elucidated. We assessed cerebrospinal beta-amyloid(1-42) and tau in 20 mild-to-moderate Alzheimer disease patients, 11 apolipoprotein E4-positive and 9 apolipoprotein E4-negative, over a mean time of 3.8 years (range 1-11.1 years). Over the period measured, cerebrospinal fluid beta-amyloid(1-42) levels were lower in apolipoprotein E4-positive compared to apolipoprotein E4-negative patients, and the levels decreased over time. Tau levels were stable over time and did not show an effect of apolipoprotein E allele. Although these results were based on a limited clinical sample, the further decrease in cerebrospinal fluid beta-amyloid(1-42) (i.e., more abnormal) combined with the cerebrospinal fluid tau stability over a mean period of almost four years suggests that beta-amyloid(1-42) and tau maintain their potential usefulness as diagnostic biomarkers over time. These findings should be taken into account if cerebrospinal fluid beta-amyloid(1-42) and tau are used as measures of treatment response.