DIRECT SEQUENCE ANALYSIS OF THE RETINOBLASTOMA GENE

视网膜母细胞瘤基因的直接序列分析

• 批准号: 3266028
• 负责人: DAVID W. YANDELL
• 金额: $ 9.8万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1993-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3266028
• 关键词: gene deletion mutation; gene rearrangement; genetic disorder diagnosis; genetic mapping; genetic markers; genetic regulatory element; human genetic material tag; human subject; neoplasm /cancer genetics; nucleic acid sequence; osteosarcoma; point mutation; polymerase chain reaction; restriction fragment length polymorphism; retinoblastoma; southern blotting

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.

该建议描述了酶扩增和直接DNA的使用 测序技术以鉴定人视网膜细胞瘤中的突变 基因。 这些方法将用于查找点突变或其他遗传 变化太小，无法通过常规方法检测到 南部印迹。 大约80％的致病突变 在视网膜母细胞瘤基因中发生的这种情况属于这一类，非常 这些突变知之甚少。 该项目的主要目标是 定义发生在 视网膜母细胞瘤基因。 这将通过分析从 各种零星或遗传性肿瘤，以及个体的血液 易感视网膜母细胞瘤或其他癌症。 频谱的知识 发生的突变 在此基因中，将有助于定义关键功能领域 视网膜母细胞瘤蛋白质，并将确定可能的热点以进行突变。 此外，该突变光谱将包括生发和 与体内相同基因中的体细胞突变。 因此，拟议的研究代表了无偏见的独特机会 单个人类的生发与体细胞突变的比较 轨迹。 拟议的研究的重要直接好处， 该提案的具体目的将用于应用这些技术 基于DNA的诊断和遗传咨询 视网膜母细胞瘤的非遗传形式。 因为这个基因是特征的 通过高新的突变率，“链接标记”或基于RFLP的诊断是 通常无效，因为以前没有 疾病。 我们认为，建立常规协议 鉴定该复杂基因中的诊断点突变 在基于DNA的诊断中迈出了重要的一步 视网膜母细胞瘤，可以作为诊断其他遗传的模型 具有高新突变率的疾病。 最后，提议扩展 这些研究以检查视网膜细胞瘤的点突变的作用 具有非典型，低渗透遗传性的几个家庭的基因 视网膜母细胞瘤和患有乳腺癌高风险的家庭。 这是 认为这些是出色的候选综合症 视网膜母细胞瘤基因的突变和鉴定 这两个群体中的诱发突变将引起极大的兴趣。 我们 相信拟议的研究不仅将具有特定的直接 与视网膜细胞瘤诊断有关的好处，但广泛 与与癌变和人类的起源相关的问题 遗传疾病。