IDENTIFICATION OF A DNA MARKER LINKED TO CYSTIC FIBROSIS

我们研究计划的长期目标是确定基本的 囊性纤维化(CF)中的缺陷。此应用程序中描述的方法 最初将绕过对CF中指定缺陷的搜索。相反，我们的 其具体目的是确定一种与这种疾病密切相关的DNA标记 轨迹。 这项研究利用了人类普遍存在的DNA序列异质性 人口。因为这些遗传序列多态很方便 用限制性内切酶检测，可变长度的 从酶消化产生的特定DNA片段已被用作 遗传分析中的标记。通过遵循多态的 CF患者家系的DNA标记，有统计方法可用 以确定这种疾病是否与该标志物有关。 对分布在整个基因组上的DNA标记的系统筛选 (不包括性染色体)应该导致一个这样的发现 与Cf基因紧密连锁的标记。这一方法预计将产生 阳性结果是因为它是为了检查患者的基因组而设计的 而不是直接测量参数，这可能是由于次要的 基本缺陷的表现形式。根据人类基因组的大小 和重组频率，估计大约有150 可能需要400个标记才能定位一个未知的基因座。不过，这个 仅设置要分析的探测数量的上限。事实上, 筛查后发现一种与亨廷顿病密切相关的标记 只有12个随机标记。 为了继续我们的研究，有两个或更多儿童的两代家庭 已被选为筛选DNA标记的小组。DNA样本是 从每个家庭成员建立的淋巴母细胞系制备， 用适当的限制性内切酶消化，用琼脂糖分级 凝胶电泳，转移到DNA结合膜上并进行杂交 用放射性标记的DNA探针。限制片段长度 然后，放射自显影揭示了基因的多态现象。为了便于分析 以及我们开发的数据存储计算机辅助系统。 Cf连锁DNA标记的发现可能会提供一个机会 用于携带者检测和产前诊断。然而，更重要的是， 该标记最终应能鉴定出Cf基因。 确定CF的基本生化缺陷是进行 这种疾病的有效治疗方法的发展。

The long term objective of our research program is to define the basic defect in Cystic Fibrosis(CF). The approach described in this application will initially bypass the search for a specifid defect in CF. Instead, our specific aim is to identify a DNA marker closely linked to the disease locus. The study exploits the DNA sequence heterogeneity prevalent in the human population. Since these hereditary sequence polymorphisms are conveniently detected by using restriction endonucleases, the variable lengths of specifc DNA fragments generated from enzyme digestions have been used as markers in genetic analysis. By following the inheritance of a polymorphic DNA marker in families with CF patients, statistical methods are available to determine whether the disease is linked to the marker. A systematic screening of DNA markers distributed over the entire genome (excluding the sex chromosomes) should lead to the discovery of one such marker closely linked to the CF gene. This approach is expected to yield positive results because it is designed to examine the patient's genome directly rather than measuring parameters which could be due to secondary manifestation of the basic defect. Based on the size of the human genome and recombination frequencies, it has been estimated that approximately 150 to 400 markers mikght be required to map an unknown locus. However, this just sets the upper limit of the number of probes to be analyzed. In fact, a marker closely linked to Huntington Disease was found after screening only 12 random markers. To pursue our study, two-generation families with two or more CF children have been chosen as panels for screening DNA markers. DNA samples are prepared from lymphoblast cell lines established from each family member, digested with appropriate restriction enzymes, size-fractionated by agarose gel electrophoresis, transferred to DNA-binding membranes and hybridized with radioactively labelled DNA probes. The restriction fragment length polymorphisms are then revelaed by autoradiography. To facilitate analysis and storage of data we have developed computer-assisted systems. The discovery of a CF-linked DNA marker will likely provide an opportunity for carrier detection and prenatal diagnosis. More importantly, however, the marker should eventually permit identification of the CF gene. Identification of the basic biochemical defect in CF is a prerequisite for the development of effective therapy for this disorder.