BIOCHEMICAL GENETICS OF CHILDHOOD IMMUNODEFICIENCY

遗传性腺苷脱氨酶(ADA)缺乏是导致 严重的联合免疫缺陷疾病，一种致命的儿童疾病。 受影响儿童的细胞ADA缺乏率不到1%。有3个 长期目标：1)克隆、定位和测序正常人ADA 基因，2)比较核苷酸序列和组织模式 ADA缺陷儿童的ADA基因突变与正常，以及3)到 从分子水平解释细胞ADA缺乏症 ADA基因的结构和表达对结构和/或变化的影响 ADA信使核糖核酸和蛋白含量。 来自19个不同ADA缺乏症儿童的细胞系。这个 这些株系在体外表达酶缺乏症，很容易代表 突变的ADA蛋白、mRNAs和基因组DNA的可用资源。研究 突变的ADA蛋白质和mRNAs已经证明这些线代表一个 ADA基因存在大量不同的突变。全长c DNA 对正常人的ADA基因进行了克隆和测序。正常和 选定的突变ADA基因将被克隆到噬菌体载体中，并将被 通过与ADA cDNA探针杂交鉴定。正常的ADA基因 将通过Sanger程序和外显子的组织进行测序， 内含子和侧翼序列将被定义。一套限制条件 代表不同内含子、外显子和侧翼区的片段 将被准备和克隆。这些将作为序列的探针 代表正常基因的不同区域，这将是有用的 信使核糖核酸前体和突变基因的表征试剂。艾达 来自ADA缺陷细胞的mRNAs将通过斑点杂交进行定量，大小由 Northern凝胶电泳法，并用S1核酸酶技术定位。 使用在mRNA中识别的缺陷作为可能的突变位置的指南， 克隆的突变基因组DNA或克隆的cDNA的适当区域将是 测序，并与正常进行比较。各种技术，包括在 体外转录、翻译和转基因将提供额外的 正常和突变ADA基因的功能特征。有一个 这些研究将很有可能确定分子缺陷在 导致致命遗传病的一系列突变等位基因。这个 信息也与ADA缺乏症的产前诊断有关 以及将正常的ADA基因转移到 有缺陷的电池。

Hereditary lack of adenosine deaminase (ADA) is the most common cause of severe combined immunodeficiency disease, a lethal childhood disorder. Cells from affected children have less than 1% ADA deficiency. There are 3 long-term objectives: 1) to clone, map, and sequence the normal human ADA gene, 2) to compare nucleotide sequences and patterns of organization of mutant ADA genes from ADA deficient children with the normal, and 3) to explain cellular ADA deficiency in molecular terms which link changes in the structure and expression of the ADA gene to changes in structure and/or amounts of ADA mRNA and protein. Cell lines are available from 19 different ADA deficient children. The lines express the enzyme deficiency in vitro and represent readily available soures of mutant ADA proteins, mRNAs, and genomic DNAs. Studies of the mutant ADA proteins and mRNAs have proved that the lines represent a large number of different mutations in the ADA gene. The full length cDNA to normal human ADA mRNA has been cloned and sequenced. Normal and selected mutant ADA genes will be cloned in phage vectors and will be identified by hybridization to the ADA cDNA probe. The normal ADA gene will be sequenced by the Sanger procedure and the organization of exons, introns, and flanking sequences will be defined. A set of restriction fragments representing the separate introns, exons, and flanking regions will be prepared and cloned. These will serve as probes for sequences representing different regions of the normal gene and will be useful reagents for characterization of mRNA precursors and mutant genes. ADA mRNAs from ADA deficient cells will be quantitated by dot blot, sized by Northern gel electrophoresis, and mapped by the S1 nuclease technique. Using defects identified in mRNAs as a guide to probable sites of mutation, appropriate regions of cloned mutant genomic DNAs or cloned cDNAs will be sequenced and compared to normal. A variety of techniques including in vitro transcription, translation, and transfection will provide additional functional characterization of normal and mutant ADA genes. There is a high probability that these studies will identify the molecular defects in a series of mutant alleles responsible for a fatal hereditary disease. The information is also relevant to the prenatal diagnosis of ADA deficiency and to the design of procedures for transfer of normal ADA genes to defective cells.