HIGH THROUGHPUT DETECTION OF GENETIC MUTATIONS AS BIOMARKERS OF FUT

癌症是一种涉及体细胞突变连续积累的遗传疾病。遗传损伤 导致这些突变的突变可以发生在基因水平（例如点突变）或染色体水平（例如， 非整倍性、易位）。已经开发了许多生物标志物来检测早期突变和 致癌物质暴露对人类染色体的影响。从历史上看，生物标志物往往测量 替代基因的突变，如HPRT，或使用细胞遗传学来评估染色体畸变（CA）。 这些生物标志物因广泛的致癌暴露而升高，CA已被证明是 在前瞻性流行病学研究中预测未来的癌症风险。然而，他们确实有几个 缺点在于替代基因不在疾病的因果途径上， 需要制备中期细胞涂片，并且它们的分析耗时且昂贵。新 因此，迫切需要癌症早期效应的生物标志物。我们建议开发生物标志物， 通过产生高通量单细胞PCR检测关键基因突变对血癌的早期影响 与白血病和淋巴瘤相关的基因或区域。这种方法将利用最近的进展 在微流体和芯片实验室技术，使单细胞遗传分析（SCGA）的可行性， 高通量规模。具体地说，我们计划开发用于执行高通量单克隆抗体的方法。 模板复制PCR扩增在纳升乳液液滴中，然后应用这些方法， 高通量SCGA方法检测血液癌症中重要突变的低频率， 如易位t（14;18）、5 q31缺失以及NFtAS和NPM 1突变。我们将测试新的SCGA是否 方法可以检测暴露于烷化剂和苯代谢物的细胞中的突变， 暴露在苯中这些研究将是提供革命性新检测方法的第一步 用于检测健康个体中与血癌风险相关的基因突变。等 生物标志物将有助于白血病和淋巴瘤的流行病学研究，这些疾病具有长潜伏期 这些措施包括在怀孕期间提供避孕药具，并为有风险的个人提供早期发现工具。高通量 我们建议开发的检测方法应适用于大规模人群研究， 与现有的生物库标本一起工作

Cancer is a genetic disease involving the sequential accumulation of somatic mutations. Genetic damage leading to these mutations can occur at the level of the gene, (e.g. point mutations) or the chromosome (e.g. aneuploidy, translocations). Numerous biomarkers have been developed to detect early mutational and chromosomal effects of carcinogenic exposure in humans. Historically, biomarkers have tended to measure mutations in surrogate genes, such as HPRT, or use cytogenetics to assess chromosomal aberrations (CA). These biomarkers are elevated by a wide range of carcinogenic exposures and CA have been shown to be predictive of future cancer risk in prospective epidemiology studies. They do, however, have several drawbacks in that surrogate genes are not on the causal pathway of disease and cytogenetic markers require metaphase spreads to be prepared and their analysis is time consuming and expensive. New biomarkers of early effect for cancer are therefore urgently needed. We propose to develop biomarkers of early effect for blood cancers by generating high-throughput single cell PCR assays for mutation in key genes or regions linked to leukemia and lymphoma. This approach will take advantage of recent advances in microfluidics and lab-on-a-chip technologies that make single cell genetic analysis (SCGA) feasible on a high-throughput scale. Specifically we plan to develop methods for performing high throughput single template copy PCR amplification in nanoliter emulsion droplets and then apply these methods to develop high-throughput SCGA methods to detect low frequencies of mutations of importance in blood cancers, such as translocation t(14;18), deletion of 5q31 and mutations in NFtAS and NPM1. We will test if the new SCGA methods can detect mutations in cells exposed to alkylating agents and benzene metabolites and humans exposed to benzene. The studies proposed will be a first step towards providing revolutionary new assays for the detection of genetic mutations of relevance to blood cancer risk in healthy individuals. Such biomarkers will be useful in epidemiological studies of leukemia and lymphoma, which have long latency periods, as well as providing tools for early detection for those individuals at risk. The high-throughput detection methods we propose to develop should be applicable to large human population studies and will work with existing biobanked specimens