RETINOBLASTOMA DIAGNOSIS USING OLIGONUCLEOTIDE PROBES

这项提议描述了一种新的快速方法 人DNA碱基对杂合性鉴定 关于已建立的酶DNA扩增技术， 双脱氧测序和等位基因特异性探针杂交。 潜在地，筛选区域中的所有多态位置都可以是 使用这种方法检测到的，并且该方法可以应用于 任何克隆的基因座。相比之下，这些多态基因中只有不到15% 可以使用现有的基于限制性内切酶的酶来检测位点 技巧。第一个主要目标是使用这一战略来 在人类基因组中识别多个多态遗传标记 视网膜母细胞瘤基因定位，并将这些标记用于基于探针的 易患视网膜母细胞瘤的诊断和遗传风险评估 家人。包括一些现有限制所在的族 片段长度多态不具有信息性，因此 目前还不可能对携带者状态进行分子诊断。 识别更多的多态标记也很重要 因为它将减少因以下原因而导致误诊的可能性 在这个大基因(大于180kb)中进行基因内重组， 当诊断是基于单个标记物时，这是一个重大风险。一个 第二个主要目标是研究遗传机制。 导致隐性肿瘤的体细胞表达- 在携带者中易患等位基因。先前在体外进行的研究 系统提示等位基因丢失，通常会扩展到很大 染色体区域，是主要的机制 这种隐性基因在杂合基因座上的表达。这 将在两个相似的遗传基因座上进行研究，其中一个在自然环境下 活体发生条件(视网膜母细胞瘤部位，研究于 肿瘤)，第二个是人类的人工杂合性基因座 体外研究的细胞系。在这两个系统中，多个链接 基因座内部和两侧的多态标记将是 这些标记的分离将被测量到 确定这些的频率、范围和分子性质 等位基因丢失事件。在两个地点自发发生的事件 将被分析和比较。使用这种方法，它将是 不仅可以确定发生的显著机制 在体内，但体外模型的准确性。它也将是 有可能在体外检查常用药物的效果 治疗肿瘤，希望治疗特别有可能 导致隐性癌基因的表达可以鉴定出来。 这种治疗可能对以下个人特别有害： 具有患上恶性疾病的遗传倾向。

This proposal describes a novel methodology for the rapid identification of base-pair heterozygosity in human DNA, based on established techniques of enzymatic DNA amplification, dideoxy sequencing, and allele-specific probe hybridization. Potentially, all polymorphic sites in a screened region can be detected using this approach, and the method can be applied at any cloned locus. In contrast, less than 15% of these polymorphic sites are detectable using existing restriction enzyme-based techniques. The first major objective is to use this strategy to identify multiple polymorphic genetic markers at the human retinoblastoma locus, and to use these markers for probe-based diagnosis and genetic risk assessment in retinoblastoma-prone families. Included are some families in which existing restriction fragment length polymorphisms are uninformative, and hence molecular diagnosis of carrier status is not currently possible. Identification of additional polymorphic markers is also important in that it will reduce the potential for misdiagnosis resulting from intragenic recombination in this large (greater than 180kb), gene, a significant risk when diagnosis is based on a single marker. A second major objective is to investigate the genetic mechanisms that lead to somatic expression of the recessive tumor- predisposing allele in carriers. Previous studies in an in vitro system suggest that allele loss, often extending over large chromosomal regions, is the predominant mechanism for expression of such a recessive gene at a heterozygous locus. This will be studied at two analogous genetic loci, one under naturally occurring conditions in vivo (the retinoblastoma locus, studied in tumors), the second an artificially heterozygous locus in a human cell line studied in vitro. In both systems, multiple linked polymorphic markers within and flanking the loci will be identified, and segregation of these markers will be measured to determine the frequency, extent, and molecular nature of these allele loss events. Spontaneously occurring events at both loci will be analysed and compared. Using this approach, it will be possible to determine not only the salient mechanisms that occur in vivo, but the accuracy of the in vitro model. It will also be possible to examine in vitro the effects of agents commonly used to treat tumors, in the hope that treatments especially likely to lead to the expression of recessive oncogenes can be identified. Such treatments may be particularly harmful to individuals that carry a hereditary predisposition to malignancy.