HIGH THROUGHPUT DETECTION OF GENETIC MUTATIONS AS BIOMARKERS OF FUT

作为 FUT 生物标志物的基因突变的高通量检测

基本信息

批准号：
7342283
负责人：
RICHARD A MATHIES
金额：
$ 39.12万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-15 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7342283
关键词：
5q31 7q22 Alkylating Agents Aneuploidy Benzene Biological Assay Biological Markers Blood Cells Cell Line Cell Separation Cells Chemicals Chromosome Structures Chromosome abnormality Chromosomes Cytogenetics Detection Development Disease Disease Pathway Early Diagnosis Emulsions Epidemiologic Studies Event Flow Cytometry Fluorescence Frequencies Future Gene Mutation Genes Genetic Genomics Genotype Goals Hematopoietic Neoplasms Hereditary Disease Human Hypoxanthine Phosphoribosyltransferase Individual Label Link Malignant Neoplasms Measures Metaphase Spread Methods Microfluidics Modeling Monitor Mutation N-ras Genes NPM1 gene NRAS gene Normal Cell Numbers Plasmids Point Mutation Polymerase Chain Reaction Production Range Risk Solutions Somatic Mutation Specimen Stem cells Technology Testing Time Work cancer risk epidemiology study genetic analysis human population study leukemia leukemia/lymphoma micro-total analysis system multiplex detection nanolitre nucleophosmin p21 N-Ras Protein prospective research study single molecule size tool

项目摘要

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

癌症是一种遗传疾病，涉及体细胞突变的顺序积累。遗传损害导致这些突变可能发生在基因水平（例如点突变）或染色体（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性暴露在人类中的染色体作用。从历史上看，生物标志物倾向于测量替代基因（例如HPRT）中的突变，或使用细胞遗传学评估染色体畸变（CA）。这些生物标志物被广泛的致癌暴露升高，CA已被证明是预测前瞻性流行病学研究中未来的癌症风险。但是，他们确实有几个该替代基因的缺点不在疾病和细胞遗传学标记的因果途径上需要中期利差要准备，他们的分析既耗时又昂贵。新的因此，迫切需要对癌症早期作用的生物标志物。我们建议开发通过产生高通量单细胞PCR分析以在钥匙中突变，对血液癌的早期作用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步在使单细胞遗传分析（SCGA）上可行的微流体和实验室芯片技术中高通量量表。具体而言，我们计划开发执行高吞吐量的方法模板复制纳米级乳液中的PCR扩增，然后应用这些方法开发高通量SCGA方法检测血液癌中重要性突变的低频，例如作为易位t（14; 18），5q31的缺失以及NFTA和NPM1中的突变。我们将测试新的SCGA 方法可以检测暴露于烷基化剂和苯代谢产物和人类的细胞中的突变暴露于苯。提出的研究将是提供革命性新测定的第一步为了检测健康个体中与血液癌风险相关的基因突变。这样的生物标志物将在白血病和淋巴瘤的流行病学研究中有用，这些研究长期存在时期，并为那些处于危险的人提供早期检测的工具。高通量我们建议开发的检测方法应适用于大型人口研究，将与现有的生物循环标本一起工作