IDENTIFICATION OF A DNA MARKER LINKED TO CYSTIC FIBROSIS

我们的研究计划的长期目标是确定基本的 囊性纤维化（CF）。 本申请中描述的方法 最初将绕过CF中特定缺陷的搜索。 而我们 具体目标是确定与疾病密切相关的DNA标记 基因座 这项研究利用了人类中普遍存在的DNA序列异质性， 人口 由于这些遗传序列多态性是方便的， 通过使用限制性内切酶检测，可变长度的 由酶消化产生特异性DNA片段已被用作 遗传分析中的标记。 通过遵循多态 CF家系DNA标志物的研究，统计学方法可用 以确定疾病是否与标记物有关。 分布于整个基因组的DNA标记的系统筛选 （不包括性染色体）应该导致发现这样一个 与CF基因紧密连锁的标记。 这种方法预计将产生 积极的结果，因为它的目的是检查病人的基因组 而不是直接测量参数，这可能是由于次级 基本缺陷的表现。 根据人类基因组的大小 和重组频率，据估计，大约150 可能需要400个标记来绘制一个未知的基因座。 但这 仅设置要分析的探针数量的上限。 事实上， 筛选后发现了一个与亨廷顿病密切相关的标记物 只有12个随机标记 为了继续我们的研究，有两个或两个以上CF孩子的两代家庭 作为筛选DNA标记的样本。 DNA样品 由从每个家族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，用琼脂糖进行大小分级 凝胶电泳，转移至DNA结合膜并杂交 放射性标记的DNA探针。 限制性片段长度 然后通过放射自显影揭示多态性。 以便于分析 和存储数据，我们开发了计算机辅助系统。 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， 该标记最终应允许鉴定CF基因。 确定CF中的基本生化缺陷是 对这种疾病的有效治疗的发展。

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.