DIRECT SEQUENCE ANALYSIS OF THE RETINOBLASTOMA GENE

本提案描述了酶扩增和直接DNA的使用。 利用测序技术鉴定人视网膜母细胞瘤的突变 吉恩。这些方法将被用来寻找点突变或其他基因 通过常规方法检测不到的微小变化，例如 南方印迹。大约80%的致病突变 发生在视网膜母细胞瘤基因中的基因属于这一类，并且非常 人们对这些突变知之甚少。该项目的一个主要目标是 为了定义发生在人类基因组中的致癌点突变的谱 视网膜母细胞瘤基因。这将通过分析来自 各种散发性或遗传性肿瘤，来自个体的血液 易患视网膜母细胞瘤或其他癌症。关于光谱的知识 所发生的突变 将有助于定义该基因的关键功能结构域 视网膜母细胞瘤蛋白，并将确定可能的突变热点。 此外，这一突变谱将包括生殖细胞和 与在体内发生的相同基因的体细胞点突变。 因此，拟议的研究为不偏不倚的 单个人的生发突变和体细胞突变的比较 轨迹。拟议研究的一个重要的直接好处，以及 该提案的具体目标是将这些技术应用于 遗传性和遗传性疾病的DNA诊断和遗传咨询 非遗传性视网膜母细胞瘤。因为这个基因的特征是 由于新的突变率很高，“连锁标记”或基于RFLP的诊断 通常是无效的，因为以前没有家族病史 疾病。我们认为，为例行程序建立礼仪 该复杂基因诊断点突变的鉴定 代表着在基于DNA的诊断方面向前迈出的重要一步 视网膜母细胞瘤，可作为其他基因诊断的模型 新变异率高的疾病。最后，建议延长 这些研究旨在检测视网膜母细胞瘤的点突变的作用 几个非典型低外显性遗传性家系的基因研究 视网膜母细胞瘤和乳腺癌高危家庭。它是 认为这些是治疗不寻常点的极好的候选综合征 视网膜母细胞瘤基因突变及其鉴定 这两个群体的易感突变都会引起人们的极大兴趣。我们 相信拟议的研究不仅会有具体的直接 与视网膜母细胞瘤诊断相关的益处，但范围广泛 与癌症发生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.