LINKAGE STUDIES IN NEUROFIBROMATOSIS

Von Recklinghausen神经纤维瘤病（NF 1）是最常见的 人类常染色体显性遗传疾病，其特征在于 乳头状斑，神经纤维瘤，虹膜的Lisch结节，以及各种 其他表现形式。 这个地方是世界上 记录的自发突变率约为10-4/等位基因/代。 在提交我们的原始报告后的短短三年里， 建议在NF 1中启动连锁研究，该基因已被 利用RFLP标记定位到染色体的近端长臂 17，通过我们集团和其他人的努力。 我们现在提出更多的分子方法， NF 1基因的克隆和鉴定。具体地说， 我们打算完善21个NF 1家族的连锁分析， 使用多点方法，并构建有序的遗传图谱， 小于1 cM的分辨率， 使用聚合酶链反应扩增标记基因座 （PCR）。 我们还将构建一个完整的物理地图近端 使用脉冲场凝胶电泳和含有17 q的文库， 包括后部限制性位点（所谓的 链接库）。 除了允许连接的部分 物理图谱，这些也提供了进入染色体的入口点 跳跃图书馆 重要的是，一组强大的体细胞 混合体可用于允许构建和映射这样的 将克隆连接到17号染色体的所需区域。 这种“反向遗传学”方法的一个主要问题是如何 识别基因本身。 幸运的是，我们有可用的手机 来自两名携带平衡易位的NF 1患者的细胞系 涉及17 q11。 NF 1基因很可能是 被这些易位所破坏，所以识别和 断裂点的克隆应该允许NF 1基因的克隆 本身 这应该是非常可以实现的使用物理 映射方法结合染色体跳跃。 一旦断点被克隆，NF 1转录本将被克隆。 通过筛选合适的cDNA文库鉴定， 组织分布研究。 NF 1的遗传基础是 通过分析该位点及其mRNA产物， 受影响的个人。 最后，我们将尝试定义 正常基因的功能，通过DNA转移实验， 培养的细胞系和转基因小鼠。

Von Recklinghausen neurofibromatosis (NF1), one of the most common human autosomal dominant conditions, is characterized by cafe-au- lait spots, neurofibromas, Lisch nodules of the iris, and a variety of other manifestations. This locus has one of the highest spontaneous mutation rates recorded, about 10-4/allele/generation. In the three short years since the submission of our original proposal to initiate linkage studies in NF1, the gene has been mapped using RFLP markers to the proximal long arm of chromosome 17, through the efforts of our group and others. We now propose additional molecular approaches which should lead to the cloning and characterization of the NF1 gene. Specifically, we intend to refine the linkage analysis of our 21 NF1 families using multipoint methods, and to construct ordered genetic maps of less than 1 cM resolution by subjecting individual sperm to amplification of marker loci using the polymerase chain reaction (PCR). We will also construct a complete physical map of proximal 17q using pulsed field gel electrophoresis and libraries containing DNA fragments which include a rear restriction site (so-called linking libraries). Besides allowing the connection of parts of the physical map, these also provide entry points into chromosome jumping libraries. Importantly, a powerful set of somatic cell hybrids is available to allow the construction and mapping of such linking clones to the desired region of chromosome 17. A major question with this "reverse genetics" approach is how to identify the gene itself. Fortunately, we have available cell lines from two NF1 patients who carry balanced translocations involving 17q11. It is highly likely that the NF1 gene is disrupted by these translocations, so that identification and cloning of the breakpoints should allow cloning of the NF1 gene itself. This should be eminently achievable using the physical mapping approach combined with chromosome jumping. Once the breakpoints are cloned, the NF1 transcript will be identified by screening of appropriate cDNA libraries, and its tissue distribution studied. The genetic basis of NF1 will be investigated by analysis of this locus and its mRNA product in affected individuals. Finally, we will attempt to define the function of the normal gene by DNA transfer experiments into cultured cell lines and transgenic mice.