RETINOBLASTOMA DIAGNOSIS USING OLIGONUCLEOTIDE PROBES

该提案描述了一种新的方法， 人类DNA中碱基对杂合性鉴定 基于已建立的酶促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性位点都可以是 使用这种方法检测，并且该方法可以应用于 任何克隆的基因座 相比之下，这些多态性的不到15%， 使用现有的基于限制性内切酶的 技术. 第一个主要目标是利用这一战略， 在人类中鉴定多个多态性遗传标记， 视网膜母细胞瘤位点，并使用这些标记物进行基于探针的 视网膜母细胞瘤倾向的诊断和遗传风险评估 家庭 包括一些家庭，其中现有的限制 片段长度多态性没有信息，因此 目前尚不可能对携带者状态进行分子诊断。 鉴定额外的多态性标记也很重要 因为它将减少由以下原因导致的误诊的可能性： 在这个大的（大于180kb）基因中的基因内重组， 当诊断基于单一标记时，这是一个重大风险。 一 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 携带者中的易感等位基因。 先前的体外研究 系统表明，等位基因丢失，往往延伸到大 染色体区域，是主要的机制， 在杂合基因座上这种隐性基因的表达。 这 将在两个类似的遗传位点进行研究，一个在自然条件下， 体内发生的条件（视网膜母细胞瘤位点，在 第二个是人类中的人工杂合基因座， 体外研究的细胞系。 在这两个系统中， 基因座内和侧翼的多态性标记将是 这些标记的分离将被测量， 确定这些的频率，程度和分子性质 等位基因丢失事件。 在两个位点自发发生的事件 将进行分析和比较。 使用这种方法，它将 不仅可以确定发生的突出机制， 但体外模型的准确性。 还将 可以在体外检查常用药物的作用， 来治疗肿瘤，希望这种治疗方法特别有可能 导致隐性癌基因表达的基因可以被识别。 这种治疗可能对以下个体特别有害： 有遗传性的恶性倾向。

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.