DIRECT SEQUENCE ANALYSIS OF THE RETINOBLASTOMA GENE

该建议描述了酶扩增和直接DNA扩增的使用。 测序技术用于鉴定人视网膜母细胞瘤中的突变 基因 这些方法将用于发现点突变或其他遗传 太小而不能用常规方法检测的变化， Southern印迹。 大约80%的致病突变 发生在视网膜母细胞瘤基因中的基因就属于这一类， 人们对这些突变知之甚少。 该项目的主要目标是 以确定发生在肿瘤细胞中的致癌点突变谱， 视网膜母细胞瘤基因 这将是 通过分析DNA， 各种散发性或遗传性肿瘤，以及来自个体血液的 易患视网膜母细胞瘤或其他癌症。 光谱知识 发生的突变 将有助于确定关键的功能域的基因， 视网膜母细胞瘤蛋白，并将确定可能的突变热点。 此外，该突变谱将包括germ和 体细胞点突变与体内发生的点突变相同。 因此，拟议的研究代表了一个独特的机会， 一个人的基因点突变与体细胞点突变的比较 基因座 拟议研究的一个重要直接好处是， 建议的具体目标，将这些技术的应用， 基于DNA的遗传和遗传咨询 非遗传性视网膜母细胞瘤 因为这个基因的特征是 通过高的新突变率，“连锁标记”或基于RFLP的诊断， 通常无效，因为没有以前的家族史， 疾病 我们认为建立常规的协议 鉴定该复杂基因中的诊断性点突变 代表了基于DNA的诊断的重要一步， 视网膜母细胞瘤，并可作为诊断其他遗传性肿瘤的模型。 新突变率高的疾病 最后，建议扩大 这些研究旨在检测视网膜母细胞瘤点突变的作用， 在几个非典型、低等位基因遗传的家系中， 视网膜母细胞瘤和乳腺癌高风险家庭。 是 认为这些是不寻常点的优秀候选综合征， 视网膜母细胞瘤基因的突变以及对视网膜母细胞瘤基因的识别 任何一组中的诱发突变都将引起极大的兴趣。 我们 我认为，这些研究不仅有具体的指导意义， 与视网膜母细胞瘤的诊断相关的益处，但广泛 与致癌作用和人类起源有关的问题 遗传病

This proposal describes the use of enzymatic amplification and direct DNA sequencing techniques to identify mutations in the human retinoblastoma gene. These methods will be used to find point mutations or other genetic changes that are too small to be detected by conventional methods such as Southern blotting. Approximately 80% of the disease-causing mutations that occur in the retinoblastoma gene fall into this category and very little is known about these mutations. A primary goal of the project is to define the spectrum of oncogenic point mutations that occur in the retinoblastoma gene. This will be accomplished by analyzing DNA from various sporadic or hereditary tumors, and from the blood of individuals predisposed to retinoblastoma or other cancers. Knowledge of the spectrum of mutations that occur in this gene will help define critical functional domains of the retinoblastoma protein and will identify possible hotspots for mutation. In addition, this mutational spectrum will include both germinal and somatic point mutations in the same gene as they have occurred in vivo. Hence the proposed studies represent a unique opportunity for an unbiased comparison of germinal versus somatic point mutations at a single human locus. An important direct benefit of the proposed studies, and a specific aim of the proposal, will be application of these techniques for DNA-based diagnosis and genetic counseling of both hereditary and nonhereditary forms of retinoblastoma. Because this gene is characterized by a high new mutation rate, "linked marker" or RFLP-based diagnosis are often ineffective because there is no previous family history of the disease. We believe that establishment of protocols for the routine identification of diagnostic point mutations in this complex gene represents an important step forward in DNA-based diagnosis of retinoblastoma, and may serve as a model for diagnosis of other genetic disorders with high new mutation rates. Finally, it is proposed to extend these studies to examine the role of point mutations of the retinoblastoma gene in several families with atypical, low-penetrance hereditary retinoblastoma and in families at high risk for breast cancer. It is argued that these are excellent candidate syndromes for unusual point mutations of the retinoblastoma gene and that identification of predisposing mutations in either group would be of great interest. We believe that the proposed studies will have not only specific direct benefits related to the diagnosis of retinoblastoma but are broadly relevant to questions elated to carcinogenesis and to the origins of human genetic disease.